Thiolesters and uses thereof

ABSTRACT

This invention pertains to the discovery of a novel family of thiolesters and uses thereof. Also provided for are viricidal compounds and pharmaceutical formulations comprising these novel thiolesters. The invention also provides thiolester-inactivated viruses and thiolester-complexed viral proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/701,451, filed May 16, 2001 now U.S. Pat. No. 6,706,729, which claimsthe benefit of Application No. 60/089,842, filed Jun. 19, 1998, whichare herein incorporated by reference in their entirety for all purposes.

FIELD OF THE INVENTION

This invention pertains to the field of virology and anti-viraltherapeutics. In particular, this invention pertains to the discovery ofa novel family of thiolesters and uses thereof.

BACKGROUND OF THE INVENTION

Viruses, especially retroviruses such as HIV, can become rapidlyresistant to drugs used to treat the infection. This extremeadaptability of retroviruses is due to the high error rate of thereverse transcriptase enzyme responsible for transcribing its RNAgenome. HIV is an example of such a hyper-mutable virus. It has divergedinto two major species, HIV-1 and HIV-2, each of which has many clades,subtypes and drug resistant variations.

Strategies for coping with emergence of viral drug-resistant strainsinclude combination drug therapies (Lange (1996) AIDS 10 Suppl1:S27-S30). Drugs against different viral proteins and drugs againstmultiple sites on the same protein are commonly used as a strategy toovercome the adaptability of the virus. Combination therapies forretroviruses, using, e.g., protease inhibitors and nucleoside analogues,such as AZT, ddI, ddC and d4T, can become ineffectual; the virusdevelops complete resistance in a relatively short period of time (Birch(1998) AIDS 12:680-681; Roberts (1998) AIDS 12:453-460; Yang (1997)Leukemia 11 Suppl 3:89-92; Demeter (1997) J. Acquir. Immune Defic.Syndr. Hum. Retrovirol. 14(2):136-144; Kuritzkes (1996) AIDS 10 Suppl5:S27-S31). Furthermore, no effective anti-retroviral vaccine iscurrently available (Bolognesi (1998) Nature 391:638-639; Bangham (1997)Lancet 350:1617-1621).

The HIV-1 caused AIDS epidemic began about 18 years ago. Since then thenumber of new cases have increased over time. By the end of 1994,1,025,073 AIDS cases had been reported to the WHO, with a 20% increasein the number of cases since December, 1993 (Galli (1995) Q. J. Nucl.Med. 39:147-155). By the year 2000, the WHO predicts that there will be30 to 40 million cumulative HIV-1 infections in the world (Stoneburner(1994) Acta Paediatr. Suppl. 400:1-4). Thus, there exists a great needfor compounds effective against retroviruses such as HIV-1. The presentinvention fulfills these and other needs.

SUMMARY OF THE INVENTION

The invention provides a novel genus of compositions comprising athiolester having a chemical structure selected from the groupconsisting of:

a thiolester having a formula selected from the group consisting ofTemplate I and Template II, wherein Template I and Template II have thestructures

wherein X is a member selected from the group consisting of alkyl,substituted alkyl, aryl, and substituted aryl groups; R₁ is —Y-Z-,

wherein Y is selected from the group consisting of —(CH₂)_(m)—, whereinm is an integer from 1 to 6,

where Z is selected from the group consisting of dialkyl or aryl oralkylaryl sulfonium (Z1), trialkyl or aryl or alkylaryl ammonium (Z2),trialkyl or aryl or alkylaryl phosphonium (Z3), or pyridinio (Z4) havingthe structure

wherein R₁₂, R₁₃, R₁₄, R₁₅, and R₁₆, are members independently selectedfrom the group consisting of H—, —C(═O)NH₂, and substituted carboxamidegroups, or,

R₁ is selected from the group consisting of alkyl, substituted alkyl,-aryl, substituted aryl, -arylalkyl, -Ph-CH₃, arylalkoxy, -Ph-OCH₃,nitroaryl, -Ph-NO₂, and —(CH₂)_(n)—X groups, where X is a halogen, and nis an integer from 1 to 6;

R₂ is selected from the group consisting of —H, —CH₃, —C(═O)NH₂ and—C(═O)OCH₃ groups;

R₃, R₄ and R₅ are members independently selected from the groupconsisting of H, a halogen, —NO₂, —C(═O)ONH₂, and —C(═O)OCH₃ groups;

R₆ is selected from the group consisting of —H, alkyl, —CH₃, substitutedalkyl, aryl, substituted aryl, and arylalkyl groups;

R₇, R₈, R₉, R₁₀ and R₁₁ are H except either R₇, R₈, or R₉ can be(O═S═O)-G′ wherein G′ is selected from the group consisting of —NH₂,—NH-alkyl, —NH-aryl, —NH-acyl, aryl-NH₂, nitroaryl, aryl-NH-acyl, andaryl-NH-alkyl groups;

a thiolester having a formula selected from the group consisting ofTemplate III and Template IV, wherein Template III and Template IV havethe structures

wherein G is selected from the group consisting of alkyl, substitutedalkyl, aryl, substituted aryl, and alkylaryl groups,

R₆ is selected from the group consisting of —H, alkyl, —CH₃, substitutedalkyl, aryl, substituted aryl, and arylalkyl groups;

R₇, R₈, R₉, R₁₀ and R₁₁ are H except either R₇, R₈, or R₉ can be(O═S═O)-G′ wherein G′ is selected from the group consisting of —NH₂,—NH-alkyl, —NH-aryl, —NH-acyl, aryl-NH₂, nitroaryl, aryl-NH-acyl, andaryl-NH-alkyl groups;

a thiolester having a formula selected from the group consisting ofTemplate V and Template VI, wherein Template V and Template VI have thestructures

wherein n is any integer, R₂ through R₅, R₆, and R₁₂ through R₁₆ aredefined as above, Y is as defined above, R₁₇ is —H or —CH₃, R₁₈ is —H,—CH₃, alkyl, aryl, arylalkyl, or an amino acid side chain, wherein thestereochemical configuration about the carbon atom to which R₁₈ isattached may be R or S, R₁₉ is —OH, —NH₂, N-substituted amide nitrogen,or an ester group (—OR);

a thiolester having a formula of Template VII with the structure

Template VII

wherein R₀ is any substituted or unsubstituted aryl or heteroaryl ringsystem attached directly to the sulfur atom,

Y and R₁₂ through R₁₆ are defined as above; and

a pyridinioalkanoyl thiolester having a formula

wherein m is an integer from 1 to 6, n is 0 or an integer from 1 to 6,and R is selected from the group consisting of alkyl, substituted alkly,aryl, substituted aryl, akylaryl, carboxamide, carboxamido, substitutedcarboxamide, substituted carboxamido, —NH₂ groups, and substituted —NH₂groups.

In alternative embodiments, in the thiolester, X is a member selectedfrom the group consisting of —(CH₂)m- , wherein m is an integer 1 to 6,and —CH₂(C═O)NH—; R₁ is selected from the group consisting of —CH₃,—(CH₂)_(n)—CH₃, —CH(CH₃)₂, —C(CH₃)₃, —(CH₂)_(n)—Cl, —(CH₂)_(n)—Br, and—(CH₂)_(n)—I groups; G is selected from the group consisting of—CH(CH₃)₂, —C(CH₃)₃; and 2,6-dimethyl phenyl groups; and, thepyridinioalkanoyl thiolester R group is selected from the groupconsisting of —NHC(═O)CH₃, —C₆H₄NO₂, —C₆H₄NHSO₂CH₂C₆H₄NO₂ and—C₆H₄NHCOCH₃ groups. The pyridinioalkanoyl thiolester can have astructure wherein m is the integer 4 and n=0 or the integer 1 or 2.

In one embodiment, the thiolester of the invention is capable ofdissociating a metal ion from a zinc finger in vitro. In anotherembodiment the thiolester of the invention has antiviral activity.

The invention provides a method for dissociating a metal ion from a zincfinger-containing protein, the method comprising the step of contactingsaid zinc finger with a thiolester of the invention. In alternativeembodiments, the metal ion is a zinc ion; the zinc finger comprises aviral protein; the viral protein is a nucleocapsid protein, a Gagprotein, or a Gag-Pol protein; and, the zinc finger-containing proteinis incorporated into an intact virus.

In one embodiment, in the method for dissociating a metal ion from azinc finger-containing protein, the contacting of said virus with saidcompound is performed in vitro. The contacting of said virus with saidcompound can also be performed in vivo. The zinc finger can comprises aretroviral protein derived from a avian sarcoma and leukosis retroviralgroup, a mammalian B-type retroviral group, a human T cell leukemia andbovine leukemia retroviral group, a D-type retroviral group, a murineleukemia-related group, or a lentivirus group. The retroviral proteincan be from an HIV-1, an HIV-2, an SIV, a BIV, an EIAV, a Visna, a CaEV,an HTLV-1, a BLV, an MPMV, an MMTV, an RSV, an MuLV, a FeLV, a BaEV, oran SSV retrovirus. This method can further comprising detecting thedissociation of said metal ion from the zinc finger of said viralprotein. The detection of the dissociation of said metal ion from thezinc finger can be carried out using a method selected from the groupconsisting of capillary electrophoresis, immunoblotting, NuclearMagnetic Resonance (NMR), high pressure liquid chromatography (HPLC),detecting release of radioactive zinc-65, detecting fluorescence, anddetecting gel mobility shift.

The invention also provides a method for inactivating a virus, saidmethod comprising contacting said virus with a compound of theinvention, wherein contacting said virus with the compound inactivatesthe virus. In this method, the compound can dissociates a zinc ion froma zinc finger. The virus can be a retrovirus derived from a aviansarcoma and leukosis retroviral group, a mammalian B-type retroviralgroup, a human T cell leukemia and bovine leukemia retroviral group, aD-type retroviral group, a murine leukemia-related group, or alentivirus group. The retrovirus can be an HIV-1, an HIV-2, an SIV, aBIV, an EIAV, a Visna, a CaEV, an HTLV-1, a BLV, an MPMV, an MMTV, anRSV, an MuLV, a FeLV, a BaEV, or an SSV retrovirus.

In the method for inactivating a virus, the contacting of the virus withthe compound can be performed in vivo. In this embodiment, the compoundcan be administered to inhibit the transmission of the virus. Thecompound can be administered intra-vaginally or intra-rectally toinhibit the transmission of the virus. The compound can be administeredto a human as a pharmaceutical formulation. The compound can beadministered to an animal as a veterinary pharmaceutical formulation.The method can further comprises contacting the virus with anon-thiolester anti-retroviral agent. The anti-retroviral agent can be anucleotide analogue or a protease inhibitor. The nucleotide analogue canbe AZT, ddCTP or DDI.

In the method for inactivating a virus, the contacting of the virus withthe compound can be performed in vitro. In this embodiment of themethod, the contacting of the retrovirus with the compound can beperformed in a blood product, blood plasma, nutrient media, protein, apharmaceutical, a cosmetic, a sperm or oocyte preparation, cells, cellcultures, bacteria, viruses, food or drink.

The invention also provides an isolated and inactivated virus, whereinthe virus is inactivated by a method comprising contacting said viruswith a thiolester of the invention, wherein contacting the virus withthe thiolester inactivates the virus. The isolated and inactivated viruscan further comprise a vaccine formulation. The isolated and inactivatedvirus can be a retrovirus derived from a avian sarcoma and leukosisretroviral group, a mammalian B-type retroviral group, a human T cellleukemia and bovine leukemia retroviral group, a D-type retroviralgroup, a murine leukemia-related group, or a lentivirus group. The viruscan be an HIV-1, an HIV-2, an SIV, a BIV, an EIAV, a Visna, a CaEV, anHTLV-1, a BLV, an MPMV, an MMTV, an RSV, an MuLV, a FeLV, a BaEV, or anSSV retrovirus.

The invention also provides a method of selecting a compound capable ofdissociating a metal ion chelated with a zinc finger of a viral protein,said method comprising: contacting the zinc finger with a thiolester,and detecting the dissociation of said metal ion from the zinc finger ofsaid viral protein. In this method, the metal ion can be a zinc ion. Inthis method, the detection of the dissociation of said metal ion fromthe zinc finger can be carried out using capillary electrophoresis,immune-blotting, Nuclear Magnetic Resonance (NMR), high pressure liquidchromatography (HPLC), detecting release of radioactive zinc-65,detecting fluorescence, or detecting gel mobility shift.

The invention also provides a kit for selecting a compound capable ofdissociating a metal ion from a zinc finger of a viral protein, the kitcomprising a retroviral protein and instructions for detecting thedissociation of said metal ion from the viral protein, the instructionscomprising directions for the selection of a thiolester of theinvention. In the kit, the viral protein can be supplied with a zinc ionchelated with the zinc finger of said viral protein. The viral proteincan be incorporated in an intact retrovirus. The zinc finger can bederived from a avian sarcoma and leukosis retroviral group, a mammalianB-type retroviral group, a human T cell leukemia and bovine leukemiaretroviral group, a D-type retroviral group, a murine leukemia-relatedgroup, or a lentivirus group. The zinc finger can be derived from anHIV-1, an HIV-2, an SIV, a BIV, an EIAV, a Visna, a CaEV, an HTLV-1, aBLV, an MPMV, an MMTV, an RSV, an MuLV, a FeLV, a BaEV, or an SSVretrovirus. In the kit, the instructions can be are directed todetecting the dissociation of said metal ion from said protein usingcapillary electrophoresis, immune-blotting, Nuclear Magnetic Resonance(NMR), high pressure liquid chromatography (HPLC), detecting release ofradioactive zinc-65, detecting fluorescence or detecting a gel mobilityshift.

The invention also provides a viricidal composition comprising athiolester of the invention. In one embodiment, the viricidalcomposition further comprises blood plasma, nutrient media, protein, apharmaceutical, a cosmetic, a sperm or oocyte preparation, cells, cellcultures, bacteria, viruses, food or drink.

The invention also provides a pharmaceutical formulation comprising athiolester of the invention. The pharmaceutical formulation can furthercomprise a pharmaceutical excipient.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification and claims.

All publications, electronic databases, patents and patent applicationscited herein are hereby expressly incorporated by reference for allpurposes.

DETAILED DESCRIPTION

The efficacy of most antiviral agents is limited because it is commonthat, under selection pressure, viruses mutate to drug-resistantstrains. Development of drug resistance is a survival strategyparticularly pronounced amongst retroviruses because of their ability torapidly mutate. Viral structures necessary for viability and growth aregood drug targets because their inactivation cannot be easily overcomeby mutation. Viral structures essential for replication and viabilityare good targets for drug development. The utility of these targets canbe further enhanced if the structures are mutationally intolerant.Furthermore, these structures maybe conserved and/or maintained betweenvirus families, groups or genuses.

The invention provides a novel genus of thiolester compositions. Thesethiolesters are capable of inactivating viruses by a variety ofmechanisms, particularly by complexing with metal ion-complexing zincfingers. In a preferred embodiment, the thiolester compositions of theinvention inactivate retroviruses. Typically, this inactivation iseffected when the thiolester contacts the virus' nucleocapsid, or otherzinc finger containing, proteins. An important aspect of these novelcompositions is that they are not effected (i.e., their activity is notgreatly diminished in vivo) by the reducing environment of biologicalfluids. Thus, they are important therapeutic reagents in the treatmentof viral, especially retroviral, agents. The viricidal activity of thecompositions of the invention are also useful in in vitro applications,such as e.g., making killed viruses to used as, e.g., reagents orvaccines, and as sterilizing reagents.

A “zinc finger” motif is a highly conserved and essential structurefound in many viruses, especially retroviruses. The Gag and Gag-Polproteins in the Retroviridae, except for Spumaviruses, contain a highlyconserved zinc finger motif (CCHC) within the nucleocapsid p7 (NCp7)protein portion of the polyprotein (see definitions, below). Theabsolute conservation of the metal chelating cysteine and histidineresidues along with other residues of the protein and its inparticipation in essential functions during early and late virusreplication identifies this feature as an antiviral target. Mutations ofthe chelating residues in the zinc fingers yield a non-infectious virus.Because zinc fingers are identical in most retroviruses, reagents ableto inhibit its function have the potential of being broad spectrumanti-viral therapeutic drugs.

HIV-1's nucleocapsid (NC) protein, NCp7, contains two zinc fingersseparated by only seven amino acids (Henderson (1992) J. Virol.66:1856). Both fingers are essential for infectivity (Aldovini (1990) J.Virol. 64:1920; Gorelick (1990) J. Virol. 64:3207). Thus, HIV-1nucleocapsid is a particularly vulnerable target for zinc fingerinactivating reagents. All evidence points toward complete conservationof the chelating residues and some other key residues within the finger.Mutation of any of these residues results in loss or severe compromiseof virus infectivity. Even mutations which maintain metal ion chelatingproperties of the finger (CCHC to CCHH or CCCC) result in loss ofinfectivity. Thus, there is no known evidence for a mutational pathwayof single or multiple mutations leading to restoration of proteinactivity.

Various C-nitroso compounds and disulfide-containing compounds, such ascystamine, thiamine disulfide, and disulfiram, can oxidize zinc fingercysteine thiolates and induce intra- and inter-molecular disulfidecross-linking, see, e.g., McDonnell (1997) J. Med. Chem. 40:1969-1976;Rice (1997) Nature Medicine 3:341-345; Rice (1997) Antimicrob. Agentsand Chemotherapy 41:419-426; Rice (1996) J. Med. Chem. 39:3606-3616;Rice (1996) Science 270:1194-1197; Rice (1993) Proc. Natl. Acad. Sci.USA 90:9721-9724; Rice (1993) Nature 361:473-475. See also Henderson, etal., WO 96/09406. Cysteine thiols in each of the two zinc fingers arerapidly attacked by reagents such as Cu⁺², Fe⁺³, C-nitroso compounds,disulfides, maleimides, alpha-halogenated ketones and nitric oxidederivatives, with simultaneous loss of the native protein structure. Forexample, treatment of intact HIV-1 with an oxidizing agent, such as3-nitrosobenzamide, a C-nitroso compound, induces disulfide linkage ofthe nucleocapsid protein and inactivates viral infectivity throughoxidation of the zinc fingers (Rice (1993) Nature 361:473; Rice (1993)Proc. Natl. Acad. Sci. USA 90:9721-9724). C-nitroso compounds can alsoinactivate eukaryotic CCHC zinc finger containing poly(ADP-ribose)polymerase (Buki (1991) FEBS Letters 290:181). However, these compoundstend to be toxic, have poor solubility and bioavailability, and arereduced and inactivated in biological solutions.

The novel thiolesters of the invention interact with the zinc finger viatheir thiolester moiety rather than an electrophilic S—S moiety. Theresultant thiolesters lack S—S electrophilic moieties. Less nucleophilicgroups are used to target zinc finger motifs. As a result, they havegreatly enhanced properties as compared to many disulfide reagents. Theyhave reduced cellular toxicity. They have enhanced antiviral activityand better reactivity with zinc finger moieties, particularly, the zincfinger on HIV-1's NCp7 nucleocapsid protein. The thiolesters of theinvention have enhanced aqueous solubilities. They maintain zinc fingerreactivity in the presence of reducing agents. The combination ofimproved characteristics, especially resistance to reduction in abiological solution, in the thiolesters of the invention clearlyenhances their use in in vitro, in vivo and therapeutic applications.

Definitions

To facilitate understanding the invention, a number of terms are definedbelow.

As used herein, the term “alkyl” is used to refer to a branched orunbranched, saturated or unsaturated, monovalent hydrocarbon radicalhaving from 1-30 carbons and preferably, from 4-20 carbons and morepreferably from 6-18 carbons. When the alkyl group has from 1-6 carbonatoms, it is referred to as a “lower alkyl.” Suitable alkyl radicalsinclude, for example, structures containing one or more methylene,methine and/or methyne groups. Branched structures have a branchingmotif similar to i-propyl, t-butyl, i-butyl, 2-ethylpropyl, etc. As usedherein, the term encompasses “substituted alkyls.” “Substituted alkyl”refers to alkyl as just described including one or more functionalgroups such as lower alkyl, aryl, acyl, halogen (i.e., alkylhalos, e.g.,CF3), hydroxy, amino, alkoxy, alkylamino, acylamino, thioamido, acyloxy,aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo, both saturated andunsaturated cyclic hydrocarbons, heterocycles and the like. These groupsmay be attached to any carbon of the alkyl moiety. Additionally, thesegroups may be pendent from, or integral to, the alkyl chain.

The term “alkoxy” is used herein to refer to the COR group, where R is alower alkyl, substituted lower alkyl, aryl, substituted aryl, arylalkylor substituted arylalkyl wherein the alkyl, aryl, substituted aryl,arylalkyl and substituted arylalkyl groups are as described herein.Suitable alkoxy radicals include, for example, methoxy, ethoxy, phenoxy,substituted phenoxy, benzyloxy, phenethyloxy, t-butoxy, etc.

The term “alkylamino” denotes secondary and tertiary amines wherein thealkyl groups may be either the same or different and are as describedherein for “alkyl groups.”

The term “aryl” is used herein to refer to an aromatic substituent whichmay be a single aromatic ring or multiple aromatic rings which are fusedtogether, linked covalently, or linked to a common group such as amethylene or ethylene moiety. The common linking group may also be acarbonyl as in benzophenone. The aromatic ring(s) may include phenyl,naphthyl, biphenyl, diphenylmethyl and benzophenone among others. Theterm “aryl” encompasses “arylalkyl.” “Substituted aryl” refers to arylas just described including one or more functional groups such as loweralkyl, acyl, halogen, alkylhalos (e.g. CF₃), hydroxy, amino, alkoxy,alkylamino, acylamino, acyloxy, phenoxy, mercapto and both saturated andunsaturated cyclic hydrocarbons which are fused to the aromatic ring(s),linked covalently or linked to a common group such as a methylene orethylene moiety. The linking group may also be a carbonyl such as incyclohexyl phenyl ketone. The term “substituted aryl” encompasses“substituted arylalkyl.”

The term “arylalkyl” is used herein to refer to a subset of “aryl” inwhich the aryl group is further attached to an alkyl group, as definedherein.

“Contacting” refers to the act of bringing components of a reaction intoadequate proximity such that the reaction can occur. More particularly,as used herein, the term “contacting” can be used interchangeably withthe following: combined with, added to, mixed with, passed over, flowedover, etc.

As used herein, the term “Gag-Pol protein” refers to the polyproteintranslation product of HIV-1 or other retroviruses, as described, e.g.,by Fehrmann (1997) Virology 235:352359; Jacks (1988) Nature 331:280-283.The “Gag protein” is processed by a viral protease to yield mature viralproteins, see, e.g., Humphrey (1997) Antimicrob. Agents Chemother.41:1017-1023; Karacostas (1993) Virology 193:661-671.

The term “halogen” is used herein to refer to fluorine, bromine,chlorine and iodine atoms.

As used herein, “isolated,” when referring to a molecule or composition,such as, for example, a thiolester of the invention, athiolester-complexed polypeptide or virus, or a thiolester-inactivatedvirus, means that the molecule or composition is separated from at leastone other compound, such as a protein, other nucleic acids (e.g., RNAs),or other contaminants with which it is associated in vivo or in itsnaturally occurring state. Thus, a compound, polypeptide or virion isconsidered isolated when it has been isolated from any other componentwith which it is naturally associated, e.g., cell membrane, as in a cellextract, serum, and the like. An isolated composition can, however, alsobe substantially pure. An isolated composition can be in a homogeneousstate and can be in a dry or an aqueous solution. Purity and homogeneitycan be determined, for example, using analytical chemistry techniquessuch as polyacrylamide gel electrophoresis (SDS-PAGE) or highperformance liquid chromatography (HPLC).

As used herein, the term “nucleocapsid protein” or “NC protein” refersto the retroviral nucleocapsid protein, which is an integral part of thevirion nucleocapsid, where it coats the dimeric RNA genome, as describedby, e.g., Huang (1997) J. Virol. 71:4378-4384; Lapadat-Tapoisky (1997)J. Mol. Biol. 268:250-260. HIV-1's nucleocapsid protein is termed“NCp7,” see also Demene (1994) Biochemistry 33:11707-11716.

The term “retrovirus” as used herein refers to viruses of theRetroviridae family, which typically have ssRNA transcribed by reversetranscriptase, as defined by, e.g., P. K. Vogt, “Historical introductionto the general properties of retroviruses,” in Retroviruses, eds. J. M.Coffin, S. H. Hughes and H. E. Varmus, Cold Spring Harbor LaboratoryPress, 1997, pp 1-26; Murphy et al. (eds.) Archives ofVirology/Supplement 10, 586 pp (1995) Springer Verlag, Wien, N.Y.; andthe web site for the Committee on International Taxonomy of Viruses,Virology Division of the International Union of Microbiology Society athttp://www.ncbi.nlm.nih.gov/ICTV/ for viral classification and taxonomy.Retroviridae family members containing zinc finger motif-containingpolypeptides and whose replication can be inhibited by the thiolestersof the invention include avian sarcoma and leukosis retroviruses(alpharetroviruses), mammalian B-type retroviruses (betaretrovirus)(e.g., mouse mammary tumor virus), human T cell leukemia and bovineleukemia retroviruses (deltaretroviruses) (e.g., human T-lymphotropicvirus 1), murine leukemia-related group (gammaretroviruses), D-typeretroviruses (epsilonretrovirus) (e.g., Mason-Pfizer monkey virus), andLentiviruses. Lentiviruses include bovine, equine, feline,ovine/caprine, and primate lentivirus groups, such as humanimmunodeficiency virus 1 (HIV-1). Examples of particular species ofviruses whose replicative capacity is affected by the thiolesters of theinvention include HIV-1, HIV-2, SIV, BIV, EIAV, Visna, CaEV, HTLV-1,BLV, MPMV, MMTV, RSV, MuLV, FeLV, BaEV, and SSV retrovirus.

As used herein, the terms “thiolester” and “thioester” may be usedinterchangeably, and they refer to a chemical structure, G-S—(C═O)-G′,wherein G and G′ represent any two groupings of atoms; and any chemicalstructure consisting of an oxygen-based carbonyl group linked directlyto a sulfur atom in the −2 oxidation state. The carbon and sulfur atoms,in turn, are linked to any two groupings of atoms; thus, G-S—(C═O)-G′.

As used herein, the term “zinc finger” refers to a polypeptide motifconsisting of cysteines and/or histidines that coordinate metal ionsgiving rise to structures involved in protein/nucleic acid and/orprotein/protein interactions. The thiolesters of the invention arecapable of dissociating metal ions from a zinc finger in vitro.Typically, the metal ion is a divalent cation, such as zinc or cadmium.A zinc finger motif-containing protein is commonly a highly conservedand essential structure in viruses. Zinc finger motifs are found inhuman papilloma virus (HPV), particularly, HPV E6 and E7 proteins (see,e.g., Ullman (1996) Biochem J. 319:229-239), influenza virus (see, e.g.,Nasser (1996) J. Virol. 70:8639-8644). In most subfamilies ofRetroviridae, including avian sarcoma and leukosis retroviruses,mammalian B-type retroviruses, human T cell leukemia and bovine leukemiaretroviruses, D-type retroviruses, and Lentiviruses, the invariable zincfinger motif is the most highly conserved structure. Retroviralnucleocapsid, Gag and Gag-Pol proteins have zinc finger motifs. Inretroviruses, the zinc finger motif typically consists of 14 aminoacids, with four residues being invariant: Cys(X)2Cys(X)₄His(X)₄ Cys andthus is referred to as a “CCHC zinc finger” (Henderson (1981) J. Biol.Chem. 256:8400). It chelates zinc through its histidine imidazole andcysteine thiolates (Berg (1986) Science 232:485; Bess (1992) J. Virol.66:840; Chance (1992) Proc. Natl. Acad. Sci. U.S.A. 89:10041; South(1990) Adv. Inorg. Biochem. 8:199; South (1990) Biochem. Pharmacol.40:123-129). CCHC zinc fingers perform essential functions in retroviralinfectivity, such as packaging genomic RNA. They are also essential forearly events in virus infection.

As used herein, the term “capable of dissociating a metal ion from azinc finger in vitro or has antiviral activity” means a thiolester iswithin the scope of the invention if, using an in. vitro assay, severalof which are described herein, it is capable of dissociating a metal ionfrom a zinc finger to any degree. Detecting the dissociation of a metalion from a zinc finger can be carried out using, e.g., capillaryelectrophoresis, immunoblotting, Nuclear Magnetic Resonance (NMR), highpressure liquid chromatography (HPLC), detecting release of radioactivezinc-65, detecting fluorescence, and detecting gel mobility shift. Theterm also means that a thiolester is within the scope of the inventionif it displays any antiviral activity in any assay, e.g., the XTTcytoprotection assay described herein. For example, a thiolester withany degree of measurable antiviral activity is within the scope of theinvention even if no metal ion dissociation is detectable.

As used herein, the terms “inhibit the transmission of the virus” and“antiviral activity” means the ability of a thiolester to negativelyeffect viral replicative capacity in any way. Such inhibition oftransmission, e.g., loss in replicative capacity, can be measured usingany means known in the art. For example, a thiolester of the inventionis inhibiting the transmission of the virus (having antiviral activity)if it diminishes a virus' ability to produce progeny, (when in the formof a virion) fuse with a cell, enter a cell, bud from a cell, surviveintracellularly or extracellularly, reverse transcribe its RNA genome,translate viral proteins, process polyproteins with proteases, effectintracellular assembly of viral components into a capsid, and the like.The ability of a thiolester of the invention to inhibit the transmissionof a virus is not limited by any chemical or biological mechanism orpathway. A thiolester can inhibit the transmission (decrease replicativecapacity) of a virus by, e.g., binding to a nucleocapsid protein, suchas NCp7; prevent binding of NCp7 to viral RNA or another nucleic acid;being involved in a specific chemical attack resulting in stable adductformation; forming intracellular disulfide bonds as a result of collapseof unstable NCp7 compounds adducts; interacting with other conserved ornon-conserved residues within the NCp7 protein which results in loss offunction; and the like.

General Methods

The present invention provides a novel genus of thiolester compoundscapable of dissociating a metal ion from a zinc finger in vitro. Theskilled artisan will recognize that the thiolesters of the invention canbe synthesized using a variety of procedures and methodologies, whichare well described in the scientific and patent literature., e.g.,Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley &Sons, Inc., NY; Venuti (1989) Pharm Res. 6:867-873. The invention can bepracticed in conjunction with any method or protocol known in the art,which are well described in the scientific and patent literature.Therefore, only a few general techniques will be described prior todiscussing specific methodologies and examples relative to the novelthiolesters and methods of the invention.

All organic reagents and intermediates were obtained from Sigma/Aldrich(St Louis, Mo.) and Lancaster Synthesis, Inc. (Windham, N.H.). Solventsand others chemicals were reagent grade. Structure and composition ofall compounds were verified by ¹H NMR and EI MS, and analyzed by silicalayer TLC, eluting with methanol/acetic acid (6:4) for thiolesters,including the pyridinioalkanoyl thioester (PATE) chemotype, andchloroform/methanol (9:1) for the others.

The thiolesters of the invention are used to inactivate zinc fingercontaining retroviruses, such as HIV-1, by attacking the zinc fingersand ejecting the zinc therefrom. It will be readily apparent to those ofskill in the art that once inactivated, the retrovirus can be used, forexample, as vaccines, as prophylactics, or as components in standardELISA assays for the diagnosis of retroviral infections. Making andusing these novel compositions and methods can involve incorporating avariety of standard procedures and reagents. Kits for identifyingcompounds that can react with HIV-1 CCHC zinc fingers are also provided.In addition to the novel compositions of the invention, these kitsincorporate a variety of standard procedures and reagents.

The following discussion of the general methods which can be used inconjunction with the present invention is intended for illustrativepurposes only. Other alternative methods and embodiments will beapparent to those of skill in the art upon review of this disclosure.

Synthesis of Disulfide Benzamide Chemotype, Compound 2D

An exemplary means to synthesize compound 2D as described in Table 1(“D” designated a dimer form, or “D form” as noted in Table 1) orN,N′-(2,2′-dithiodibenzoyl)-bis-sulfacetamide, follows. The startingmaterial, 2,2′-dithiodibenzoyl chloride, was synthesized as described byKatz (1953) J. Org. Chem 18:1380-1402; Baggaley (1985) J. Med. Chem.28:1661-1667. To a solution of sulfacetamide (13 g, 60 mmol) in pyridine(300 ml) was added dropwise a solution of 2,2′-dithiodibenzoyl chloride(as 85%, 8.1 g, 20 mmol) in 1,4-dioxane (100 ml) at room temp (RT). Theclear, reddish-brown solution was stirred at RT overnight, then pouredinto vigorously stirred ethyl ether (1 L). The viscous liquidprecipitate was separated from the ether phase, dissolved inN,N-dimethylformamide (DMF, approximately 50 ml), and added dropwise to800 ml of vigorously stirred, aqueous 3 M HCl. The white precipitate wasfiltered off, washed with water and dried in vacuum. Yield, 11 g (78%).The crude product (1 g) was dissolved in hot ethanol (20 ml). The hotfiltrate was added to stirred water (200 ml). The white precipitate wasfiltered off and dried. Yield was 0.85 grams (85%) of pure 2D. ¹H NMR(DMSO-d₆), (12.08 (s, ¹H, HNSO₂), 11.04 (s, 1H, HN-Ph), 8.01 (AB q , 4H,H-Ph), 7.87 (d, 1H, J=7.6 Hz, H-Ph), 7.81 (d, 1H, J=7.8 Hz, H-Ph), 7.59(t, 1H, J=7.7 Hz, H-Ph), 7.46 (t, 1H, J=7.4 Hz, H-Ph), 1.97 (s, 3H,CH₃); EI MS m/z 699 (MH⁺); Anal. Calcd (C₃₀H₂₆N₄O₈S₄): C, 51.56; H,3.75; N, 8.02. Found: C, 51.34, H, 3.84, N, 8.05.

Synthesis of Benzoisothiazolone Chemotypes, Compounds 2B, 22, 31, 34

Exemplary means to synthesize benzoisothiazolone chemotypes, includingcompound 2B as described in Table 1 (the “B” designates the BITA, orbenzoisothiazolone form) orN-[4-(3-oxo-3H-benz[d]isothiazol-2-yl)phenylsulfonyl] acetamide,follows. Two methods were used. In one method, to a solution of compound2D (0.2 g, 0.28 mmol) in pyridine (2 ml) was added a solution ofN-bromosuccinimide (0.18 g, 1 mmol) in 1,4-dioxane (1 ml). The solutionwas stirred at RT for 3 hours and added to water (30 ml). The whiteprecipitate was collected and purified by precipitation from hot ethanoland water. Yield, 0.17 g (87%). ¹H NMR (DMSO-d₆), (12.24 (s, 1H, NH),8.14 (d, 1H, J=8.0 Hz, H-Ph), 8.10 (s, 4H, H-Ph), 8.02 (d, 1H, J=7.8 Hz,H-Ph), 7.83 (t, 1H, J=7.0 Hz, H-Ph), 7.56 (t, 1H, J=7.6 Hz, H-Ph), 2.00(s, 3H, CH₃); EI MS m/z 349 (MH⁺); Anal. Calcd (C₁₅H₁₂N₂O₄S₂): C,51.71.; H, 3.47; N, 8.04. Found: C, 51.42, H, 3.57, N, 8.04.

The second method was used to synthesize compound 2B, compound 22 BITA,compound 31 BITA and compound 34 BITA (see Table 1). To a mixture of2,2′-dithiodibenzoyl chloride (0.32 g, 0.93 mmol) in CCl₄ (10 ml) wasadded a solution of 2.5% w/v Cl₂ in CCl₄ (10 ml). The mixture wasstirred until it cleared (1 h). After filtration, the filtrate wasbubbled with N₂ for 1 h, and a solution of sulfacetamide (0.2 g, 0.93mmol) in N,N-dimethylacetamide (DMA, 4 ml) was added. The mixture wasstirred for 2 h, ethyl ether (20 ml) was added, and the precipitate wascollected and purified with hot ethanol and water. Yield for compound2B, 0.3 g (92%); the same ¹H NMR and EI MS as method A. Anal. Calcd(C₁₅H₁₂N₂O₄S₂): C, 51.71; H, 3.47; N, 8.04. Found: C, 51.34, H, 3.64, N,7.96.

Synthesis Spaced Disulfide Benzamide/Benzoisothiazolone Chemotype,Exemplary Compounds 23D, 24D and 25D

Exemplary means to synthesize compounds 23D (see Table 1)orN,N′-(2,2′-dithiodibenzoyl)-bis-4-(aminomethyl)benzene-sulfonanide, 24D,or N,N′-(2,2′-dithiodibenzoyl)-bis-4-(2-aminoethyl)benzene-sulfonamide,and 25D, orN,N′-(2,2′-dithiodibenzoyl)-bis-4-(glycinamido)benzenesulfonamide (seeTable 1) follows. These compounds were prepared, respectively, from4-(aminomethyl) benzenesulfonamide (Aldrich), 4-(2-aminoethyl)benzenesulfonamide (Aldrich), and N-glycylsulfanilamide. The lattercompound was prepared by first treating sulfanilamide with equimolaramounts of bromoacetyl bromide and pyridine in DMA and recovering thebromoacetylated derivative after adding the reaction mixture to excess0.5 M HBr. The dried, crude product was recrystallized from ethanol andconverted to N-glycylsulfanilamide via a standard Gabriel reaction(preparing the phthalimido derivative and cleaving with hydrazinehydrate). Procedures for compound 2D, outline above, were then followed.

Compound 23D: ¹H NMR (DMSO-d₆), (9.34 (t, 1H, J=6.1 Hz, NH), 7.85 (d,2H, J=8.3 Hz, H-Ph), 7.79 (d, 1H, J=7.6 Hz, H-Ph), 7.70 (d, 1H, J=8.0Hz, H-Ph), 7.59 (d, 2H, J=8.3 Hz, H-Ph), 7.51 (t, 1H, J=7.7 Hz, H-Ph),7.37 (m, 3H, NH₂ and H-Ph), 4.61 (d, 2H, J=5.9 Hz, CH₂); EI MS m/z 643(MH⁺); Anal. Calcd (C₂₈H₂₆N₄O₆S₄): C, 52.32; H, 4.08; N, 8.72. Found: C,52.10, H, 4.25, N, 8.53.

Compound 24D: ¹H NMR (DMSO-d₆), (8.81 (t, 1H, J=5.2 Hz, NH), 7.81 (d,2H, J=8.0 Hz, H-Ph), 7.66 (d, 1H, J=8.0 Hz, H-Ph), 7.61 (d, 1H, J=7.3Hz, H-Ph), 7.54-7.46 (m, 3H, H-Ph), 7.34 (m, 3H, NH₂ and H-Ph), 3.58 (q,2H, J=6.3 Hz, CH₂—N), 3.01 (t, 2H, J=7.1 Hz, CH₂-Ph); EI MS m/z 671(MH⁺); Anal. Calcd (C₃₀H₃₀N₄O₆S₄): C, 53.71; H, 4.51; N, 8.35. Found: C,53.43, H, 4.68, N, 8.33.

Compound 25D: ¹H NMR (DMSO-d₆), (10.53 (s, 1H, HN-Ph), 9.09 (t, 1H,J=5.8 Hz, HN—CH₂), 7.87-7.83 (m, 5H, H-Ph), 7.72 (d, 1H, J=7.1, H-Ph),7.55 (t, 1H, J=7.0 Hz, H-Ph), 7.39 (t, 1H, J=7.6 Hz, H-Ph), 7.31 (s, 2H,NH₂), 4.18 (d, 2H, J=3.7 Hz, CH₂); EI MS m/z 729 (MH⁺); Anal. Calcd(C₃₀H₂₈N₆O₈S₄(H₂O): C, 48.25; H, 4.05; N, 11.25. Found: C, 48.54, H,4.12, N, 11.20.

Synthesis N-Terminally Modified Aminophenyl Sulfone Chemotype, ExemplaryCompound 34D

Exemplary means to synthesize compound 34D (see Table 1) orN,N′-(2,2′-dithiodibenzoyl)-bis-4-sulfanilyl-N-[(2-nitrobenzyl)sulfonyl)]aniline,follows. Compound 26 was made in a similar manner as compound 2D,starting with 3-aminophenyl sulfone and 2,2′-dithiodibenzoyl chloride.To a solution of compound 26 (1.5 g, 1.9 mmol) in DMA (30 ml) was added(dropwise) a solution of 2-nitro-toluenesulfonyl chloride (1.4 g, 5.9mmol) in 1,4-dioxane (10 ml). The solution was stirred at RT overnightand then transferred to vigorously stirred ethyl ether (300 ml). Afterremoving the ether phase, the viscous liquid was diluted with DMF (15ml). The diluted solution was added to water (200 ml) with stirring. Thewhite precipitate was collected and purified by precipitation twice fromhot ethanol and ethyl ether. Yield compound 34D: 1.92 g (84%). ¹H NMR(DMSO-d₆), (11.01 (s, 1H, HN—SO₂), 10.74 (s, 1H, HN—CO), 8.10-7.29 (m,16H, H-Ph), 5.11 (s, 2H, CH₂); EI MS m/z 1165 (MH⁺); Anal. Calcd(C₅₂H₄₀N₆O₁₄S₆): C, 53.60; H, 3.46; N, 7.21. Found: C, 53.49, H, 3.84,N, 7.13.

Synthesis 2,3-Haloalkanoamido Benzamido Chemotype, Exemplary Compound 37

Exemplary means to synthesize compound 37 (see Table 1) orN-[2-(3-chloro-propionamido) benzoyl]sulfacetamide, follows.N-(2-nitrobenzoyl) sulfacetamide was made in a similar manner as 2D, butstarting with 2-nitrobenzoyl chloride. One gram (2.7 mmol) of thisproduct was dissolved in methanol (100 ml) at 45° C. The solution wascooled to room temp. and then bubbled with N₂ to remove air. To thesolution was added palladium, 10 wt. % on activated carbon, (0.22 g)under N₂. The mixture was bubbled with H₂ for 1.5 h and then with N₂ for0.5 h. After filtration, the filtrate was evaporated to dryness. Yield,0.84 g (93%) of white-yellow product. This 2-aminobenzamido derivativewas reacted with 3-chloropropionyl chloride under conditions similar tothose described for compound 34D, yielding compound 37. ¹H NMR(DMSO-d₆), (12.06 (s, 1H, NH—SO₂), 10.84 (s, 1H, NH-Ph), 10.32 (s, 1H,NH-Ph), 8.03-7:91 (m, 5H, H-Ph), 7.76 (d, 1H, J=7.7 Hz, H-Ph), 7.60 (t,1H, J=7.8 Hz, H-Ph), 7.32 (t, 1H, J=7.6 Hz, H-Ph), 3.87 (t, 2H, J=6.1Hz, CH₂Cl), 2.86 (t, 2H, J=6.2 Hz, CH₂), 2.00 (s, 3H, CH₃); EI MS m/z424 (MH⁺); Anal. Calcd (C₁₈H₁₈N₃O₅SCl): C, 51.00; H, 4.28; N, 9.91.Found: C, 50.85, H, 4.49, N, 9.96.

Synthesis Haloalkanoyl Thioester Chemotype, Exemplary Compound 44

Exemplary means to synthesize compound 44 (see Table 1) orN-[2-(5-bromovaleroylthio) benzoyl]sulfacetamide, follows.N-(2-mercaptobenzoylsulfacetamide was first prepared by adding to asolution of 2D (2.2 g, 3.1 mmol) in 90% DMF (20 ml),tris(2-carboxyethylphosphine hydrochloride (1 g, 3.5 mmol) andtriethylamine (0.5 ml). The solution was stirred for 1 h and then addedto 0.5 M HCl (200 ml). The precipitate was collected and dried, yielding2.3 g (95%). To a solution of this product (0.5 g, 1.4 mmol) in DMA (5ml) was added 5-bromovaleryl chloride (0.6 ml, ˜4.5 mmol) under N₂. Thesolution was stirred under N₂ for 1 h and added to ethyl ether (50 ml).After decanting the ether phase, the remaining viscous liquid wasdissolved in DMF (10 ml). The solution was added to vigorously stirredwater (100 ml). The white precipitate was filtered off and purified fromhot ethanol and water. Yield compound 44: 0.47 g (65%). ¹H NMR(DMSO-d₆), (12.05 (s, 1H, HNSO₂), 10.91 (s, 1H, HN-Ph), 7.94 (s, 4H,H-Ph), 7.74-7.61 (m, 4H, H-Ph), 3.48 (t, 2H, J=6.3 Hz, CH₂Br), 2.74 (t,2H, J=7.1 Hz, CH₂CO), 1.96 (s, 3H, CH₃), 1.90-1.64 (m, 4H, CH₂CH₂); EIMS m/z 515 (MH⁺); Anal. Calcd (C₂₀H₂₁N₂O₂S₂Br): C, 46.79; H, 4.12; N,5.46. Found: C, 47.16, H, 4.31, N, 5.60.

Synthesis of Pyridinioalkanoyl Thioester Chemotype, Exemplary Compound45

Exemplary means to synthesize compound 45 (see Table 1) orN-[2-(5-pyridinio-valeroylthio) benzoyl]sulfacetamide bromide, follows.A solution of compound 44 (0.25 g, 0.49 mmol) in pyridine (7 ml) wasstirred under N₂ overnight. Ethyl ether (80 ml) was added, and the whiteprecipitate was collected and purified from hot ethanol and ether; theprecipitate was dried in vacuum immediately. Yield compound 45, 0.21 g(72%). ¹H NMR (DMSO-d₄), (12.10 (br. s, 1H, HNSO₂),10.94 (s, 1H, HN-Ph),9.12 (d, 2H, J=6.1 Hz, 2,6-H-Py), 8.65 (t, 1H, J=7.2 Hz, 4-H-Py), 8.21(t, 2H, J=7.0 Hz, 3,5-H-Py), 7.94 (S, 4H, H-Ph), 7.74-7.58 (m, 4H,H-Ph), 4.62 (t, 2H, J=7.2 Hz, CH₂-Py), 2.79 (t, 2H, J=7.2 Hz, CH₂—CO),1.92 (m, 5H, CH₃ and CH₂), 1.57 (pentet, 2H, J=7.6 Hz, CH₂); EI MS m/z512 (M⁺); Anal. Calcd (C₂₅H₂₆N₃O₅S₂Br): C, 50.68; H, 4.42; N, 7.09.Found: C, 50.54, H, 4.68, N, 7.22.

Synthesis of Thiolesters Represented by Templates I, II and III

Exemplary means to synthesize the thiolester compounds represented byTemplates I, II and III follow. As discussed above, the skilled artisancan use any synthetic scheme or any variation to an exemplary protocolto generate a thiolester of the invention.

In templates II and IIIb, where X is —CH₂—, —CH₂CH₂— and —CH₂C(═O)—NH—,the thiolester can be generated using methods for synthesizing compounds23D, 24D and 25D, respectively, described above (see Table 1).

Where the thiolester is N-[2-(α-Pyridinio-4-toluoylthio) benzoyl]sulfacetamide chloride, a solution of N-(2-mercaptobenzoyl)sulfacetamide (see synthesis compound 44) (0.4 g) and 4-(chloromethyl)benzoyl chloride (0.6 ml) in dimethylacetamide (2 ml) was stirred undernitrogen for 1 h, and then added to ethyl ether (40 ml) with stirring.The viscous liquid precipitate was diluted with dimethylformamide (1ml), and then added to a solution of ether (40 ml) and heptane (40 ml).The viscous liquid precipitate was collected and added to pyridine (3ml). The solution was stirred at RT under Ar for 3 days, and then addedto ether (50 ml). The precipitate was collected and dried. The crudeproduct was purified on a silica gel column using isocratic elution with10% AcOH in MeOH.

Where the thiolester is N-[2-(2-(Pyridinioacetamido)benzoylthio)benzoyl]sulfacetamide chloride, an analog of compound 37 was prepared inthe same manner except that chloroacetyl chloride was substituted for3-chloropropionyl chloride. This product was dissolved in pyridine andallowed to stand at RT, and the product was worked up as described abovefor N-[2-(α-Pyridinio-4-toluoylthio)benzoyl] sulfacetamide chloride.

Where the thiolester is N-[2-(Pyridinio acetamido acetylthio)benzoyl]sulfacetamide chloride, N-(2-Mercaptobenzoyl)sulfacetamide is preparedas described for compound 44. Chloroacetylglycine may be linked to thethiol group of this product following activation of the glycine carbonylgroup via (a) a p-nitrophenyl ester derivative, or (b) an acid chlorideprepared using oxalyl chloride. Workup and subsequent reaction withpyridine is carried out as described forN-[2-(α-Pyridinio-4-toluoylthio) benzoyl]sulfacetamide chloride,described above.

Where the thiolester is N-[2-(2-(Pyridinioacetamido) isobutyrylthio)benzoyl]sulfacetamide chloride, the compound can be prepared in asimilar manner as is N-[2-(Pyridinio acetamidoacetylthio)benzoyl]sulfacetamide chloride, described above, except thatN-chloroacetyl-2-aminoisobutyric acid (prepared according to Birnbaum(1952) J. Biol. Chem. 194:455 and Ronwin (1953) J. Org. Chem. 18:127,)is substituted for chloro-acetylglycine.

Where the thiolester is N-[2-(5-Dimethylsulfoniovaleroylthio)benzoyl]sulfacetamide iodide, wherein Z=—S⁺(CH₃)₂ and Y=—(CH₂)₄—: amixture of compound 44 (1.10 g) and NaI (5 g) in acetone (99.9%, 25 ml)was stirred under nitrogen at RT overnight, and then added to water (250ml) with stirring. The white precipitate was collected and dried. Yield,1.2 g. A clear solution of this product (0.3 g) in acetonitrile (3 ml)and methyl sulfide (4 ml) was stirred at RT overnight. The clearsolution was added to ethyl ether (250 ml) with vigorous stirring. Thewhite precipitate was collected, washed with ether, and dried. Yield,0.15 g.

Where the thiolester is N-[2-(5-Triethyl ammoniovaleroylthio) benzoyl]sulfacetamide iodide, wherein Z=—N⁺(C₂H₅)₃ and Y=—(CH₂)₄—: a parallelsynthesis to N-[2-(5-Dimethylsulfoniovaleroylthio)benzoyl]sulfacetamideiodide may be conducted wherein triethylamine is substituted for methylsulfide. Refluxing may be required to finish reaction withtriethylamine.

Where the thiolester is N-[2-(5-Tri-n-butylphosphonio valeroylthio)benzoyl]sulfacetamide iodide, wherein Z=—P⁺(C₄H₉)₃ and Y=—(CH₂)₄—: aparallel synthesis to N-[2-(5-Dimethylsulfoniovaleroylthio)benzoyl]sulfacetamide iodide may be conducted whereintri-n-butylphosphine is substituted for methyl sulfide.

To synthesize a thiolester specie of Template III, where G is t-butyl, Yis —CH₂—, X is —CH₂—, the pyridine ring bears a substituted carboxamidegroup at R₈ and R₁₀, R₆ is H, R₉ is =—SO₂—NH₂, and R₇, R₈, _(R10), R₁₁is H: 4-[1-(t-Butylthiocarbonylmethyl)nicotinamidomethyl]-benzenesulfonamide chloride triethylamine (36 mmol)and nicotinoyl chloride hydrochloride (30 mmol) were added to asuspension of 4-(aminomethyl) benzenesulfonamide hydrochloride hydrate(30 mmol) in 150 ml acetonitrile. An additional 60 mmol of triethylaminewere added gradually. The precipitate of 4-(nicotinamidomethyl)benzenesulfonamide was recrystallized from a solution of 12 ml ethanolin 180 ml water. Product dried in vacuo/CaCl₂. Next,S-chloroacetyl-t-butyl mercaptan is prepared by reacting chloroacetylchloride and t-butyl mercaptan according to Dawson (1947) J. Amer. Chem.Soc. 69:1211. The thiolester is generated by stirring 10 mmol of thenicotinamide derivative with 20 mmol of the chloroacetyl thioester in125 ml acetonitrile at reflux overnight. On standing at RT, the productcrystallizes out. It may be recrystallized from an ethanol-watermixture.

In templates II and IIIb, where R₁ is alkyl, —CH₃, —(CH₂)n-CH₃,—CH(CH₃)₂, —C(CH₃)₃, -aryl, -arylalkyl, -Ph-CH₃, arylalkoxy, -Ph-OCH₃,nitroaryl, -Ph-NO₂, —(CH₂)_(n)—X where X is a halogen, —(CH₂)_(n)—Cl,—(CH₂)_(n)—Br, or —(CH₂)_(n)—I; the group ofN-(2-acylthiobenzoyl)sulfacetamides, having acyl groups with the formulaR₁—C(═O)—and R₁'s taken from these alternative R₁ structures, can beprepared as described for compound 44 and substituting R₁—C(═O)—Cl for5-bromovaleryl chloride.

Thiolesters based on Templates I and II with R2 being —CH₃ rather thanH, can be prepared in the same manner as compounds 44 and 45 bysubstituting 2,2′-dithiobis (3-methylbenzoyl chloride) for2,2′-dithiodi-benzoyl chloride in the preparation of the precursor,compound 2D. The 3-methyl derivative can be prepared according to themethod of Collman and Groh (1982) J. Amer. Chem. Soc. 104:1391- 1400.

Thiolesters based on Template I with R6 being —CH3 can be prepared asdescribed for compounds 44 and 45, except that, in place ofsulfacetamide, N-methyl-4-(4-nitrobenzenesulfonyl)aniline is used andprepared as described by Saxena (1989) Arzneim. Forsch. 39:1081-1084.

Thiolesters based on Template II with R₆ being —CH₃ can be prepared asdescribed for compounds 44 and 45, except that2-methylamino-N-(4-sulfamoylphenyl) acetamide is used and the thiolesterprepared according to Horstmann (1977) Eur. J. Med. Chem. Chim. Ther.12:387-391.

Thiolesters based on Template II with R6 being aryl or phenyl can beprepared as described for compounds 44 and 45, except that, in place ofsulfacetamide, one would use N-phenylglycyl-sulfanilic acid amide,prepared as described by Gaind, Sehgal and Ray; J. (1941) Indian Chem.Soc. 18:209.

Design and Synthesis of Thiolesters Capable of Dissociating Metal Ionsfrom Zinc Fingers

Viral zinc fingers, particularly the zinc finger in the nucleocapsidprotein of NCp7 of HIV-1, are used as models to design the novelthiolesters of the invention. NCp7 possess regions on their solventaccessible surfaces where favorable interactions with candidate ligandsmay occur. Each zinc finger has two potential high affinity bindingsites. One comprises the putative mRNA binding site near Phe17 on finger1 and Trp37 on finger 2. The other site is near the metal coordinatinghistidine in each finger, opposite the putative mRNA site. For thepurposes of designing novel thiolesters capable of dissociating metalions from zinc fingers, the ligand binding site opposite the putativemRNA binding region was considered the stronger of the two candidatebinding sites. These binding regions were probed with a range ofpossible hydrophobic and hydrophilic atom types. Those types with thestrongest possible binding interactions were selected.

Further modeling studies of the interactions of ligands (agents) withNCp7 suggested that both disulfide benzamides (DIBAs) andbenzoisothiazolone derivatives (BITAs) can interact with hydrophobicpatches on the surfaces of both retroviral Zn fingers in such a way asto orient the reactive groups of these agents into close proximity withthe nucleophilic cysteine sulfur atoms in each finger. In contrast,DIBAs failed to interact with the cysteine sulfur atoms in the Znfingers of the transcriptional factor GATA-1 due to steric exclusion(Huang (1998) J. Med. Chem. 41:1371-1381). Disulfides and theirrespective BITA derivatives were designed, synthesized and purified, andthe antiviral and in vitro NCp7 zinc ejection activities were assessed.

TABLE 1 Synthesis of Novel Disulfides and Benzoisothiazolone FormsDerived From the DIBA-1 and DIBA-2 Chemotypes

Antiviral Activity^(a) Compound R Form EC₅₀ μM IC₅₀ μM TI 1 —NH₂ ^(b) D 0.85 217   255.3  BITA — 16.2 2.

D BITA 1.5 12.6  >200     34   >133      2.7 3.

D BITA 3.8 9.3 54.1 49.5 14.2  5.3 4.

D BITA 2.9 7.1 18.3 19.2  6.3  2.7 5.

D BITA 2   6.5 46.4 50.8 23.2  7.8 6.

D BITA 2.1 7.6 52.5 56.6 25   7.4 7.

D BITA 4.2 8.8 56.7 57.4 13.5  6.5 8.

D BITA 2.1 17.8  55.5 57.6 26.2  3.3 9.

D BITA 1.6 6.9 32.2 17.1 20.1  2.5 10.

D BITA 3.4 7.8 18.6 17.3  5.5  2.2 11.

D BITA 1.9 8.7 27.6 54.6 14.5  6.3 12.

D BITA 4.2 22.6  53.8 55.3 12.8  2.4 13.

D BITA 1.6 NT^(d) 46.5 29.1 14.

D BITA 2.4 NT 46.4 19.3 15.

D BITA 0.33  NT 19.5 59.1 16.

D BITA 1.1 NT 17.8 16.2 17.

D BITA 0.72  9.2 22.2 54.7 30.8  5.9 18.

D BITA 1   NT 18   18   19.

D BITA 1.9 6.5 18.2 39.7  9.6  6.1 20.

D BITA 2.5 12.8  44.2 39.0 17.7  3.0 21.

D BITA 111 NT >316     >2.8 22.

D BITA 2.3 6.2 43   18.1 18.7  2.9 ^(a)Antiviral activity was measuredin the XTT cytoprotection assay. ^(b)Originally reported as DIBA-1^(c)Originally reported as DIBA-2 ^(d)NT designates compounds that couldnot be made in sufficient quantity and or purity to analyze

TABLE 2 Modification of the DIBA-2 Chemotype by Backbone LinkerSubstitutions

Antiviral Activity^(a) Zinc Finger Reactivity Compound R Form EC₅₀ μMIC₅₀ μM TI RFU/30 min^(b) 23. —CH₂— D 0.33 19.4 58.8 3.3 BITA 13.8 53.73.9 2.7 24. —(CH₂)₂— D 0.41 52.8 139 2.7 BITA 2 45.5 22.7 2.7 25.

D 1.6 17.6 11 7.9 ^(a)Antiviral activity was measured by the XTTcytoprotection assay ^(b)Zinc finger reactivity measured by the Trp37fluorescence assay. Results are expressed as the average decrease inrelative

TABLE 3 Optimization of the Bis(Aminophenyl)Sulfone-Based DisulfideBenzamides

Antiviral Activity^(a) Zinc Finger Reactivity Compound R Form EC₅₀ μMIC₅₀ μM TI RFU/min^(b) 26. —NH₂ D 4.3 <316 >73.5   3.3 27.

D 1.5   160 106.7  5.9 28.

D BITA 1.0 9.6     12.7   188 12.7 19.6 5.9 7.9 29.

D 3.8     79.6 20.9 6.9 30.

D 12.2    190 15.6 — 31 —NO₂ D 12.2  >316 >26     2.9 BITA — >316 — 4.132.

D 78   >316 >4   2.9 33.

D 240    >316 >1.3 1.6 34

D BITA 12.3  3.2 >316     59.5 >25.7   15.1 1.5 2 ^(a)Antiviral activitywas measured by the XTT cytoprotection assay ^(b)Zinc finger reactivitywas measured by the Trp37 fluorescence assay. Results are expressed asthe average decrease in relative

TABLE 4 3,3′ Bis(Aminophenyl) Sulfone Isomers

Zinc Finger Reactivity Antiviral Activity^(a) RFU/ Compound R EC₅₀ μMIC₅₀ μM TI min^(b) 35 —NH₂ 1 81.5 81.5 0.92 36.

0.62 5.9 95 2 ^(a)Antiviral activity was measured by the XTTcytoprotection assay ^(b)Zinc Finger reactivity measured by the Trp37fluorescence assay. Results are expressed as the average decrease inrelative fluorescence units over 30 min.

TABLE 5 Single Liganded (Monomer) Benzamides Linked to HaloalkanoylGroups via Amide or thioester Bonds

Reactivity Antiviral Activity^(a) Zinc Finger Reactivity Compound R1 R2EC₅₀ μM IC₅₀ % TI RFU/min^(b) Amide: 37.

H — >316 — 2.0 38 H

38 103 2.8 3.9 39.

H — 202 — 0.3 Thioesters: 40.

H 2.8 56.7 20.3 4.2 41.

H 2.6 45.7 16.6 2.2 42.

H 2.8 43.7 15.6 3.5 43.

H 4.2 62 14.8 9.2 44.

H 3.8 184.5 49.2 1.8 45.

H 6.2 >316 >51 3.6 ^(a)Antiviral Activity was measured by the XTTcytoprotection assay ^(b)Zinc finger reactivity measured by the Trp37fluorescence assay. Results are expressed as the average decrease inrelative fluorescence units over 30 min.

TABLE 6 Evaluation of Pyridinoalkanoyl thioesters (PATEs) and4-bromovaler- oyl Thioesters

Zinc Reactivity Antiviral Activity^(a) Finger Compound R Group EC₅₀ % μMIC₅₀ μM TI RFU/min^(b)

Compound 2 Backbone: 44. R1 38 184.5 49.2 1.8 45. R2 6.2 >316 >51 3.6

Compound 31 Backbone: 46. R1 1.6 21.7 13.6 1 47. R2 5.5 >316 >57 4.1

Compound 23 Backbone: 48. R2 1.1 55 50 1.4

Compound 34 Backbone: 49. R1 — >316 — 1.9 50. R2 2.9 >316 >109 0.86

Compound 27 Backbone: 51. R2 4.6 288 63 1.7

Compound 36 Backbone: 52. R2 4.9 205 43 1.1

Partial Structure (R2): 53. — >316 4 ^(a)Antiviral Activity was measuredby the XTT cytoprotection assay ^(b)Zinc finger reactivity measured bythe Trp37 fluorescence assay. Results are expressed as the averagedecrease in relative fluorescence units over 30 min.

TABLE 7 Antiviral Mechanism of Action Compound (I₅₀:M^(a)) Activity^(b)44 45 47 Integrase NI^(c) NI NI Reverse transcriptase rAdT NI NI NI rCdGNI NI NI Protease NI NI NI Zinc Finger Reactivityd 1.8(13.3%) 3.6(34.7%)4.1(17.9%) Attachment NI NI NI Fusion >100 77 99 ^(a)I50 Concentrationof compound inhibiting 50% of the indicated activity. ^(b)All positivecontrols for individual assays are as noted in the Experimental Section.^(c)No inhibition (NI) at a high dose test of 100 μM. dExpressed asdecrease in relative fluorescence units per 30 min. (RFU/30 MIN), withpercent total decrease in flourescence given in parenthesis.

TABLE 8 Effect of glutathione on Antiviral Activity of Selected PATEsand 5-Bromovaleroyl Thioesters Zinc Finger Reactivity^(a) CompoundGSH^(b) RFU/min % Decrease 44 − 6.5 60.1 + 2.4 28 45 − 2.6 28.2 + 3.836.9 47 − 1.6 24.3 + 1.6 21.1 PBS control − 0 0 + 1.3 7.9 ^(a)Zn ringerreactivity was measured by the Trp37 flourescence assay. Reactivity isexpressed as either the average change in fluorescence units per 30 min(RFU/min) or as the total percent decrease during a 30 min incubation (%decrease). ^(b)Compounds (2 mM) were treated for 2 h at 37° C. with 4 mMreduced glutathione. Following incubation reactions were diluted to afinal concentration of μM and activity in the Trp37 Zn ejectiondetermined as previously described.

TABLE 9 Summary of Zinc Finger Reactivity for Compounds 44, 45 and 47 U1NCp7 Virion Nucleic Acid Virucidal Inhibition of Gag Precursor Compound(Trp37) Cross-link Binding (I₅₀ μM) Activity (I₅₀ μM) p24 (EC₅₀ μM)Cross-linking 44 + −/+ 100 12.3 94 +/− 45 + +++ 1 13.2 42.2 +/− 47 + −/+100 2.1 10.7 +++

TABLE 10 Evaluation of Structural Features Comprising thePyridinoalkanoyl Side Chain of the PATEs

Antiviral Activity^(b) Zinc Finger Reactivity Compound X n R EC₅₀ % μMIC₅₀ % TI RFU/min^(b) 54 S 3 H 10.1 175 17.3 2.2 45 (parental PATE) S 4H 6.2 >316 >51 3.6 55 S 5 H 10.5 69.7 6.5 1.9 56 NH 4 H — >316 — 2.6 57S 4 Cl 54 264 4.8 0.2 ^(a)Antiviral Activity was measured by the XTTcytoprotection assay ^(b)Zinc finger reactivity measured by the Trp37fluorescence assay. Results are expressed as the average decrease inrelative fluorescence units over 30 min.

Disulfide, and where synthetically possible, the BITA forms, ofcompounds 1 through 36 were synthesized (Tables 1 to 4) and theirantiviral activity was assessed in the XTT cytoprotection assay(described below).

Essentially all compounds generated for these studies possessed somedegree of antiviral activity (XTT cytoprotection assay) and zinc fingerreactivity. Zinc finger reactivity was measured using a Zn specificfluorochrome, TSQ, N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide, assayor a Trp37 zinc finger fluorescence assay. Fluorescent measurements ofthe Trp37 residue in the C-terminal finger of recombinant HIV-1 NCp7protein were performed as described by Rice (1995) Science270:1194-1197; Rice (1997) Antimicrob. Agents Chemother. 41:419-426.Measurement of Zn ejection from both fingers was measured using theZn-selective fluorchrome probe TSQ (Molecular Probes, Eugene Oreg.) asdescribed by Rice (1996) J. Med. Chem 39:3606-3616. Measurement of theability of NCp7 to bind to a DNA oligomer, a 44 mer: GGC GAC TGG TGA GTACGC CAA AAA TTT TGA CTA GCG GAG GCT AG (SEQ ID NO:1), analogous to theHIV-1 RNA packaging site was carried out as described by Huang (1998) J.Med. Chem. 41:1371-1381, see also, Rossio (1998) HIV Pathogenesis andTreatment: Keystone Symposium on Molecular and Cellular Biology,Abstract # 4082. Briefly, 50 nM NCp7 was treated with test compounds for1 hour at RT in 10 ml buffer containing 10% glycerol and 50 mM Tris-HCl(pH 7.5). Labeled oligomer (0.1 picomole, end labeled [³²P] was added inan equal volume of buffer containing 10% glycerol, 50 mM Tris-HCl (pH7.5), 400 mM KCl and 40 mM MgCl₂. The reaction was continued for 15 minat room temperature, and a total of 5 ml (or ¼ of the total reactionvolume) was separated on nondenaturing 4.5% polyacrylamide gels in 0.5×Tris-Borate electrophoresis buffer. NCp7-oligomer complexes werevisualized by autoradiography.

Among the new disulfide-based analogs, compounds 26, 31 and 34 exhibitedno toxicity at the high test dose of 316 μM (micromolar) and exhibitedan EC₅₀ (concentration inhibiting 50% virus replication) of 4.3, 12.2and 12.3 μM, respectively. The aminophenyl substitution used to generatecompound 26 converted the ligand portion of the DIBA into a bis(4-aminophenyl) sulfone. N-acetylation of compound 26 yielded compound27 with a lower EC₅₀ (1.5 μM) but with greater toxicity (160 μM).Conversion of the bis(aminophenyl) sulfone bridge of compound 26 to asulfonamido group and changing the terminal amine to a primarysulfonamide (compound 22, Table 1) resulted in much greater cellulartoxicity (43 μM). Generally, where disulfide/BITA pairs could besynthesized, there was an average of a 4-fold (n=15, range 1.5- to8-fold) drop in the in vitro therapeutic index (IC₅₀/EC₅₀), primarilybecause of a loss in antiviral potency, i.e., increased EC₅₀ values.There was not an enhanced cellular toxicity. None of the new disulfidesor BITAs were superior to compound 1.

Molecular modeling suggested that better fits of agents onto the NCp7 Znfinger atomic surface binding domains could be achieved by the additionof methylene (compound 23) or ethylene (compound 24) spacers in thebackbones between the benzamido head group and the benzenesulfonamideligand group of compound 1 (Table 2). Antiviral activity and Zn fingerreactivity were maintained, but these modifications were associated witha 4- to 10-fold decrease in IC₅₀ (more cytotoxic) when compared to theparental compound 1. As seen in Table 2, the corresponding BITAsdemonstrated a significant loss of antiviral activity (compound 23:42-fold decrease, and compound 24: 5-fold decrease), even thoughreactivity against purified NCp7 was maintained. Further modification ofthe head group spacer by addition of a carbamyl group to compound 23(see compound 25) enhanced zinc finger reactivity (RFU/min 7.9 versus3.3), but decreased antiviral potency.

Optimization of Bis(aminophenyl) Sulfone-Based Disulfide Benzamides

Incorporation of 4,4′-bis(aminophenyl) sulfone onto the disulfide headgroup yielded compound 26 with a terminal amine as a synthetic locus forfurther modifications. Several acyl modifications generated compounds27, 28 and 29, which resulted in increased zinc finger reactivity andimproved EC₅₀. These gains were partially (compounds 27 and 29) andcompletely (compound 28) offset by decreases in the IC₅₀. Thus far,terminal extension of the ligand portion of 26 did not yield asignificantly improved antiviral agent.

The BITA derivative of compound 26 could not be produced in sufficientpurity or quantity to study its properties. However, interchanging theterminal NH₂ of 26 for an NO₂ group, producing compound 31, overcamethis constraint and allowed for the production of both the disulfide andits corresponding BITA (Table 3). The disulfide form of compound 31,like 26, was non-toxic and had equivalent NCp7 zinc finger reactivity,even though the EC₅₀ increased 2.6-fold (less effective). The BITA formof compound 31 was without antiviral activity. Exploration of ligandextensions with nitro aromatic groups led to compounds 32 to 34 thatgenerally displayed less zinc finger reactivity than the simpler nitrogroup, as in compound 31. Only the disulfide and BITA forms of compound34 showed significant antiviral activity.

Repositioning of the sulfonyl group in 3,3′-bis(aminophenyl) sulfonescreated isomers, compounds 35 and 36, Table 4. These compounds haveimproved antiviral activities (compound 26 vs. compound 35: EC₅₀ 4.3 to1 mM; 27 vs. 36: EC₅₀ 1.5 to 0.62 mM, respectively). However, zincfinger reactivity was significantly reduced (26 vs. 35: 3.3 to 0.92RFU/min; 27 vs. 36: 5.9 to 2 RFU/min, respectively). In addition, thischange decreased the IC₅₀ by at least 3-fold (greater toxicity). Thus,the 3,3′-diphenyl sulfone isomers provided no major advantages.

Conversion of the Disulfide to a Novel Thiolester

The action of disulfides on the NCp7 zinc fingers involves athiol-disulfide interchange between the agent and the CCHC cysteinesulfur atoms. The resulting covalent disulfide linkage between NCp7 andone-half of the drug (“monomeric” portion) causes disruption of the zincchelate with passive loss of Zn²⁺ ions from the NCp7 structure (see,e.g., Rice (1995) supra; Huang (1998) supra.

To covalently modify zinc finger cysteines through bonds that are morestable than would be possible using any of the compounds describedabove, novel thioethers were designed and synthesized. To create athioether link, a moderately active, SH-selective alkylating function atthe ortho or meta positions of the benzamide head group was designed.Single-liganded (monomer) benzamides were linked to potentially reactivehaloalkanoyl groups via amide or thioester bonds (see Table 5).Compounds 37 through 39 represent amide linkages to either the ortho ormeta benzamide positions. Substitution of Cl—(CH₂)₂—CO—NH— at either theortho (compound 37) or meta (compound 38) positions on. the ligand formof compound 2 generated compounds with modest zinc finger reactivity.However, neither had appreciable antiviral activity. Compound 39, havingan ortho Cl—CH₂—CO—NH—, was completely inactive. Ortho amide substitutedprobes were synthesized using several other backbones as the ligandstructure and gave equivalent results.

Ortho positioned, alkanoyl, aroyl, and haloalkanoyl thioesters weredesigned and synthesized (compounds 40 to 52, Tables 5 and 6). Synthesisof the acetyl (compound 40), benzoyl (compound 41), 4-methoxybenzoyl(compound 42) and the butyroyl (compound 43) thioesters resulted in fourcompounds with low μM EC₅₀ values and moderate to substantial zincfinger reactivity. However, due to IC₅₀ values in the 50 μM range, theirantiviral activity approximated that of the BITA derivatives (Table 1).

A new thioester was synthesized using co-haloalkanoyl groups. The ortho5-bromovaleroyl thioester derivative of compound 2, compound 44,resulted in significantly reduced cytotoxicity and moderate zinc fingeractivity. The therapeutic index was further enhanced by convertingcompound 44 to the pyridinioalkanoyl thioester (PATE) derivative,compound 45. The resulting agent demonstrated efficient zinc fingerreactivity (3.6 RFU/min), no toxicity at the high test dose of 316 μMand an EC₅₀ of less than 10 μM.

Table 6 shows that the PATEs exhibit as a chemotype more favorableantiviral and toxicity profiles than their parental compounds. The lackof toxicity displayed by the pyridiniovaleroyl thioesters is illustratedby their addition to the monobenzamide backbones of compounds 2, 31, 34,27 and 36, producing the new compounds 45, 47, 50, 51 and 52,respectively, where cellular toxicity of the parent monobenzamide islessened in all cases. Even though conversion of the compound 23backbone to the PATE chemotype, resulting in compound 48, did notgenerate selectivity indexes (TI) of the order found in the otherpyridiniovaleroyl thioesters, and failed to reduce monobenzamidetoxicity (compound 23 BITA 53.7 μM vs. compound 48 μM),pyridiniovaleroyl thioester conversion partially relieved the toxicityassociated with the disulfide form (IC₅₀ of compound 23D: 19.4 μM vs.compound 48: 55 μM). Thus, thiolesters, and PATEs in particular,represent a new chemotype which can be used as novel chemotherapeuticagents to target viral zinc fingers.

Antiviral Activity of Pyridinioalkanoyl Thioesters (PATEs)

The 5-bromovaleroyl thioester, compound 44, and two 5-pyridiniovaleroylthioesters, compounds 45and 47, were analyzed in mechanistic andtarget-based assays. These compounds did not inhibit HIV-1 integrase,reverse transcriptase or protease enzyme activities (see Table 7).Assays for activity against HIV-1 reverse transcriptase rAdT(template/primer) and rCdG (template/primer) using recombinant HIV-1reverse transcriptase (S. Hughes, ABL Basic Research NCI-FCRDC,Frederick Md.) were performed as described by Rice (1997) supra.Substrate cleavage of recombinant HIV-1 protease in the presence of testcompounds using an HPLC-based methodology with the artificial substrateAla-Ser-Glu-Asn-Try-Pro-Ile-Val-amide (Multiple Peptide Systems, SanDiego Calif.) was performed as described by Rice (1997) supra. Theability of recombinant HIV-1 integrase (S. Hughes, ABL Basic ResearchNCI-FCRDC, Frederick Md.) to carry out 3′ processing and strand transferactivities in the presence of test compounds was performed as describedby Buckheit (1994) AIDS Res. Hum. Retroviruses 10:1497-1506, and Turpin(1998) Antimicrob. Agents Chemother. 42: 487-494.

The 5-bromovaleroyl thioester compound 44 and the two5-pyridiniovaleroyl thioester compounds 45 and 47 also did not inhibitvirus attachment (cell-based p24 attachment assay) and fusion to hostcells. The cell-based p24 attachment assay was performed as described inRice (1995) Science 270:1194-1197. Viral inactivation assays werecarried out as described in Rice (1995) Science 270:1194-1197, andTurpin (1997) supra, with minor modifications. Briefly, MAGI-CCR-5 cellswere plated (4×10⁴ cells per well) in flat bottomed 2-cm well plates for24 h, after which the culture media was removed and compound-treatedvirus was added. The virus used was obtained by transient transfectionof pNL4-3 into HeLa cells yielding the replication competent NL4-3virus. Virus containing supernatants were treated for 2 h at 37° C.after which residual compound was removed by centrifugation (18,000×g, 1h, 4° C.). Virus pellets were resuspended and placed on the MAGI-CCR-5monolayers and cultured for 48 h. Monolayers were fixed and stained withX-gal solution and blue cells counted.

Determination of the effect of the test compounds on viral fusion werecarried out as follows. Briefly, HL2/3 cells stably expressing HIV-1 Tatand cell surface gp120 and MAGI-CCR-5 cells stably expressing CD4 andCCR5 on their cell surface, containing a β-galactosidase reporter geneunder the control of the HIV-1 LTR were pretreated with test compoundfor 1 h at 37° C. After incubation, the cells were mixed in a 4 to 1ratio (2×10⁵ MAGI-CCR-5 to 8×10⁵ HL2/3 cells) and co-cultured for 18 hat 37° C.; cultures were fixed and stained with X-gal solution forβ-galactosidase activity. Blue cells represented the Tat transactivationof the HV-1 LTR upon fusion of the HL2/3 and MAGI-CCR-5 cells via theCD4/CCR5 co-receptor gp120 interaction.

All three thiolester compounds 44, 45, and 47 promoted metal ion (zinc)ejection from the NCp7 protein, as assessed by the Trp37 assay,described above. These compounds interfered with, and thus could not betested in, the TSQ fluorochrome assay (see Table 9).

Interaction of zinc finger inhibitors with NCp7 can result in loss ofprotein structure and their ability to specifically bindoligonucleotides representing the HIV-1_(ψ)packaging site (Huang (1998)supra; Tummino (1997) Antimicrob. Agents Chemother. 41:394-400; South(1993) Protein Sci. 2:3-19; Dannull (1994) EMBO J. 13:1525-1533). Theability of NCp7 to specifically associate with these oligonucleotidesafter treatment of the protein with a thiolester of the invention wastested. For example, compounds 44, 45 or 47 were very effective atinhibiting Ncp7's ability to associate with target oligonucleotide, asdetermined using electrophoretic mobility shift assays (EMSA) onnondenaturing polyacrylamide gels. At 1 μM, compound 45 inhibitedcomplex formation. Compounds 44 and 47 inhibited complex formation at100 μM (see Table 9).

To determine if compound 44, 45 or 47 binding activity was specific forCCHC zinc fingers, a super gel shift EMSA after treatment of K562 cellnuclear extracts with the compounds was performed. This was followed bysuper shifting with Sp1-specific antibody. Sp1 was chosen because it isa cellular transcription factor that contains three copies of aclassical type CCHH zinc finger motif that are required for Sp1 bindingto its DNA target. The three compounds 44, 45 or 47 failed to alter thepattern of super shifting of Sp1 bound to its DNA target, indicatingthat these thiolesters show specificity for the CCHC retroviral Znfinger. Super gel shifting to determine the specificity of zinc fingerreactive compounds was carried out using an Sp1 consensus oligomer(Stratagene, La Jolla, Calif.), K562 cell nuclear extracts, andantibodies for Sp1 (Sp1[PEP2]-G) (extracts and Sp1 Abs from Santa CruzBiotechnology, Inc., Santa Cruz, Calif.). The binding reactions ofoligonucleotides with K562 nuclear extract, as well as electrophoreticconditions were carried out as described in the Gelshift Buffer Kit,Stratagene , La Jolla, Calif.

The interaction of zinc finger inhibitors, such as the thiolesters ofthe invention, with cell-free virus can result in modification ofintravirion NCp7 protein and loss of infectivity. Virion-associated NCp7proteins from cell-free HIV-1_(MN) were treated with thiolestercompounds 44 and 45. They were also evaluated by non-reducing Westernblotting using HIV-1 NCp7-specific antibodies. The PATE compound 45resulted in extensive cross-linking of NCp7, whereas its haloalkanoylthioester precursor (compound 44) was a very poor cross-linking agent.Intravirion NCp7 cross-linking by compound 45 was comparable to DIBA-1,compound 1. Cross-linking by compound 45 was due to formation ofintermolecular disulfide bonds because reduction with 2-mercaptoethanol(β-ME) completely reversed the gel retardation. Compound 47 did noteffectively initiate NCp7 cross-linking. AZT, a nucleoside reversetranscriptase inhibitor, also failed to cross-link NCp7 (see Table 9).

NCp7 cross-linking is associated with viral inactivation. Whether theability to crosslink the nucleocapsid protein, as determined by Westernblotting, correlated with the ability to inactivate the viruses wastested in a virucidal assay. Virus stocks of HIV-1_(NL4-3) wereincubated with various thiolester compounds. The virus was obtained bytransient transfection of a replication-competent pNL4-3 plasmid intoHeLa cells. Residual thiolester was removed by centrifugation. The viruspellet was resuspended in culture media and used to infect MAGI-CCR-5cells. At 48 hours post-infection, the cells were washed and stainedwith X-Gal. Compounds 44 and 45 were virucidal with I₅₀ (concentrationresulting in 50% virus inactivation) values of 12.3 μM and 13.2 μM,respectively. Although compound 47 was not a potent cross-linker ofintravirion NCp7, it was approximately 6-fold (I₅₀=2.1 μM) more potentthan compounds 44 and 45 at inactivating virus. These data suggest thatthiolesters, and compound 47, in particular, form stable adducts withthe zinc finger sulfur atoms that do not participate in the generationof intermolecular or intramolecular disulfide cross-linkages.

Analysis of 5-pyridiniovaleric acid (compound 53) without conjugation toa monobenzamide backbone showed significant reactivity in the Trp37 zincejection assay (4 RFU/min), even though it was without antiviralactivity (see Table 6). Compound 53 was further assessed for intravirioncross-linking of NCp7 and virucidal activity. Neither intravirioncross-linkage of NCp7 nor virucidal activity were detected. Thus, themonobenzamide backbones of compounds 44, 45 and 47 are essential foractivity against cell-free virions.

Thiolester compounds were also tested for their ability to. inhibitHIV-1 replication in TNFα-induced U1 and ACH-2 cells. U1 cells wereinduced with 5 ng/ml of TNFα in the presence of various concentrationsof the thiolesters 44, 45 or 47. Forty-eight hours later the cultureswere characterized for virus p24 antigen production and cell viability.All three compounds inhibited the release of HIV-1 virions (p24) from U1cells (EC50: 94, 42.2 and 10.7 μM for compounds 44, 45 and 47,respectively) (see Table 9). No cellular toxicity was evident at thehigh test dose of 100 μM. Higher dose testing (300 μM) of all threethiolesters showed no toxicity for compounds 45 and 47. Compound 44killed 60% of the cells at 200 μM. Visualization of viral proteins fromTNFα-induced U1 cells by electrophoretic separation and immunoblotting(Western blotting) revealed that compounds 45 and 47 inducedcross-linking of viral precursor polyproteins and prevented processingof those precursors. Reduction of the U1 protein preparations with β-MEshowed that both compound 45 (100 μM) and compound 47 inhibitedPr^(55gag) precursor processing. Compound 44 also induced β-MEreversible alterations in the mobility of zinc finger containing HIV-1precursor proteins at a intermediate level between compounds 45 and 47.Precursor processing evaluation performed as described by Turpin (1997)Antiviral Chem. Chemother. 8:60-69. Thus, thiolester PATEs initiateintracellular disulfide cross-linking of precursor polyproteins duringlate phase virus assembly and mediate a direct virucidal effect via anattack on the mature NCp7 retroviral zinc fingers in the cell-freevirus.

Virion cross linking was performed as described by Rice (1995) Science270:1194-1197 and Turpin (1997) Antiviral Chem. Chemother. 8:60-67.Briefly, highly purified HIV-_(1MN) (11.8 μg total protein) wasincubated for 2 hour with concentrations of compound. Virions wereconcentrated and residual compound was removed by centrifugation(18,000×g, 1 h, 4° C.). The viral pellet was solubilized in 0.5 MTris-HCl, (pH 6.8), 50% glycerol, 8% SDS and 0.4% bromophenol blue andproteins resolved by Western blotting. Western blotting to detect theexpression of HIV-1 proteins in U1 cells or virus pellets for virioncross-linking experiments was carried out as described by Turpin (1996)J. Virol. 70:6180-6189. Briefly, 50 μg of total cellular protein for U1experiments or the. total virion pellet (11.8 μg total viral protein)for NCp7 cross linking studies was resolved on 4 to 20% polyacrylamidegels in SDS with Tris-Glycine (Novex, San Diego Calif.). Samples forreduced gels were boiled for 5 min in the presence of 5% β-ME prior toloading. Resolved proteins were electroblotted on to polyvinylidenedifluoride (PVDF) membranes, and HIV-1 specific proteins were detectedusing a mixture of goat anti-HIV-1 NCp7 and p24 or anti-NCp7 for virionproteins alone (a kind gift of L. E. Henderson, AIDS Vaccine ProgramNCI-FCRDC, Frederick, Md.). Western blots were developed using standardchemiluminescence methodologies, as produced and described byDupont-NEN, Wilmington, Del.

One significant characteristic of thiolesters is their ability tomaintain zinc finger reactivity in the presence of the reducingenvironment of an in vivo biological fluid. This property can beevaluated, e.g., by testing the compound's resistance to reduction byglutathione under physiologic conditions. This resistance to reductionis a significant improvement over all disulfide containing compounds.For example, the disulfide Nature of DIBAs make them inherently unstablein reducing environments. This results in the disassociation of dimer tomonomeric forms and mixed disulfides.

The thiolester PATE compounds 44, 45 and 47 maintained the capacity toattack the cysteine thiols in the presence of a 2 molar excess ofglutathione. Thus, conversion of the disulfide to the less nucleophilic5-bromovaleroyl thioester, as in compound 44, or, 5-pyridiniovaleroylthioester, as with compounds 45 and 47, confers significant resistanceto reductive agents, thus preventing subsequent loss of zinc fingerreactivity (see Table 8).

Regarding the Table 10, the activity profile of the PATE chemotype wasdependent upon the length of the alkyl spacer between the carbonyl andpyridinio moieties (comparing Compounds 54, 45, and 55). The n=4 spacingappears to be optimal with regard to potency and toxicity. The essentialpresence of the thiolester sulfur is demonstrated by the inactiveCompound 56 where the sulfur is replaced by —NH—. Substituting achlorine for hydrogen meta to the nitrogen in the pyridinium ringsignificantly diminishes antiviral potency, possibly due to anelectrostatic interaction (repulsive) between the chloro group andresidues 39 and 49 of NCp7.

Detecting the Dissociation of a Metal Ion from a Zinc Finger Motif

The invention provides a method and kit to select compounds capable ofdissociating a divalent ion chelated with a zinc finger motif. The motifcan be isolated, or a substructure of a viral protein or a virion. Themethod includes contacting the zinc finger with a thiolester; andsubsequently detecting the dissociation of the metal ion from the zincfinger protein. The cation is commonly zinc. Any methodology known inthe art can be used to detect the dissociation of the metal ion.Exemplary means include, e.g., capillary electrophoresis,immune-blotting, Nuclear Magnetic Resonance (NMR), high pressure liquidchromatography (HPLC), detecting release of radioactive zinc-65,detecting fluorescence, or detecting gel mobility shift, and othertechniques which would be apparent to one of skill upon review of thisdisclosure. These procedures can be practiced with any protocol known inthe art, which are well described in the scientific and patentliterature. A few exemplary techniques are set forth below.

As the invention also provides a genus of novel thiolesters capable ofdissociating a metal ion from a zinc finger in vitro, detection of thedissociation of the metal ion identifies a thiolester specie within thescope of the invention. The zinc ejection assay was used as a first linescreen to identify thiolesters within the scope of the invention. Onestrategy for such screening used the XTT cytoprotection assay to monitoranti-viral activity and the Trp37 zinc ejection assay to identifythiolesters able to act at the cellular level on the NCp7 protein or itsGag or Gag-Pol precursors.

A thiolester is within the scope of the invention if it is capable ofany level of cation ejection from a zinc finger. In fact, a thiolestercapable of zinc ejection at a low rate, i.e., with slow kinetics, ispreferable for some uses, especially for certain in vivo applications.An exemplary “weak cation ejector” thiolester of the invention iscompound 50, with a zinc ejection rate of 0.86 RFU/min as measured bythe Trp37 zinc finger fluorescence assay. However, thiolesters withlower ejection rates (<0.86 RFU/min) are also within the scope of theinvention. A “high” ejection rate would be in the range of approximately8 RFU/min. Thiolesters with an antiviral activity EC₅₀ of <15 uM wereselected as preferred embodiments.

Capillary Zone Electrophoresis (CZE)

Retroviral zinc finger proteins complex with two zinc ions, each with aformal charge of ⁺2. Reagents that react with the protein and remove thezinc ions cause a change in the conformation and charge of the protein.Thus, the electrophoretic mobility of the reacted protein will differfrom the mobility of the unreacted protein. Changes in electrophoreticmobility of the protein can easily be detected by the standard techniqueof capillary zone electrophoresis (CZE). For a general description ofCZE, see, e.g., Capillary Electrophoresis, Theory and Practice (AcademicPress, Inc. Grossman and Colburn (eds.) (1992).

Generally, electrophoretic mobility of the protein (at a pH determinedby the buffer in the capillary electrophoresis tube) is used to move theretroviral protein from a fixed starting position towards one electrode.The migration rate may be monitored by UV absorption, e.g., at 215 nm.Sample tubes containing an appropriate amount of a solution comprisingthe retroviral NC protein of choice, with and without the thiolestercompound to be tested for CCHC zinc finger inactivation, are placed inan automatic sample injector. At programmed intervals, samples are drawninto the capillary tube and the UV absorption is monitored. Unmodifiedretroviral NC protein gives a sharp peak of migrating protein passingthe detector. Modifications of the protein, caused by reaction with thetest compound of choice, are revealed by a change in the electrophoreticmobility of the reacted protein.

Capillary zone electrophoresis has the advantage of simple automation,since many different samples can be loaded and analyzed in successiveruns. Each run requires about 10 minutes and each sample tube can beanalyzed multiple times. An example of a kit utilizing CZE for analysisof selected compounds to be tested for CCHC zinc finger reactivity wouldcontain about 100 micrograms (μg) of purified retroviral NC proteincomplexed with zinc in, for example, 1.0 ml of water, and could be usedfor the testing of approximately 1000 test compounds.

Release of Radioactive Zinc from Zinc-65 Labeled NCp7

Purified HIV-1 NCp7 can be reconstituted with radioactive zinc-65 with adetermined specific activity. By monitoring the release of radioactivezinc-65 caused by the reaction of a test compound with a retroviral NCprotein, it is possible to determine the reactivity of the testcompound.

A thiolester test compound can be added to a solution containing the NCprotein complexed with radioactive zinc-65. Following the reaction,protein (reacted and unreacted) can be precipitated, for example, byimmunoprecipitation or immunoadsorbtion methods using known antibodies,or by the addition of a calibrated amount of nucleic acid such that theNC protein saturates the binding sites on the nucleic acid matrix. Underconditions of low ionic strength, the saturated protein-nucleic acidcomplex forms a precipitate that can be removed by centrifugation.Alternatively, labeled nucleocapsid protein may be attached to a solidsupport, and test reagents added directly to the attached protein. Anyreactions releasing zinc from the protein can be detected by the releaseof radioactive zinc-65 into the soluble supernatant. This generalprocedure can be automated depending on the equipment available.

A kit supplying retroviral nucleocapsid protein and appropriateprecipitating agents can be used to detect the ability of test compoundsto remove zinc from the protein.

Release of Radioactive Zinc from Zinc-65 Labeled Whole Virus

Zinc is present in virus in quantities nearly stoichiometric with CCHCzinc finger arrays (Bess (1992) J. Virol. 66:840). Nearly all of thezinc is coordinated in the CCHC arrays (Summers (1992) Protein Science.1:563). Therefore, zinc released from a virus derives from zincpreviously coordinated in CCHC arrays, rather than from unrelatedproteins or other non-specific associations with the virion.

Purified virus can be produced from cells cultured in the presence ofadded zinc-65. Labeled virus can be isolated and purified by densitygradient centrifugation in the presence of added EDTA to remove anyunbound zinc. The purified virus can be any retrovirus of interestincluding, but not limited to, HIV-1, HIV-2 or SIV.

Compounds to be tested (thiolesters of the invention) can be added tothe purified radioactive virus under conditions appropriate for the testcompound of choice (Rice (1993) Nature 361:473-475). Following thereaction, removal and/or inactivation of the reagent, the virus isdisrupted by the addition of a non-ionic detergent (e.g., Triton X-100),and the viral core containing the NC protein complexed to nucleic acidis removed by centrifugation.

Radioactive zinc-65 released into the supernatant indicates that thetest compound penetrated the intact virus and disrupted the NCprotein-zinc complex. Kits to determine whether test compounds canremove retroviral NC-chelated zinc would contain, for example, intactretrovirus particles with radioactive zinc-65 incorporated into their NCproteins, appropriate reaction buffers and a non-ionic detergent.

Fluorescence-Based Detection of Zinc Dissociation from Protein

Changes in the intrinsic fluorescence of aromatic protein moieties arecommonly used to monitor a reaction which involves a change in proteinconformation. In the present invention, fluorescence can be used tomonitor the loss of metal ion from a zinc finger, e.g., the loss of zincfrom a CCHC retroviral zinc finger protein, after contact with athiolester of the invention. The intrinsic fluorescence of Trp37 in thesecond zinc finger of HIV-1 NC protein has been used to monitor nucleicacid binding and conformation of the zinc finger complex (see, Summers(1992) supra).

Zinc ejection is measured by the ability of compounds to chemicallyattack the cysteines in purified NCp7 protein resulting in a loss offluorescence due to the. movement of the tryptophan 37 residue from anexposed to an internal position on the protein. Zinc ejection ismeasured and expressed as either percent decrease in total fluorescenceor decrease in relative fluorescence units per min during a 30 min assay(RFU/min). A thiolester is considered within the scope of the inventionif any amount of zinc ejection is detected. For example, compound 50 hasa zinc ejection rate of 0.86 RFU/min.

Artificial fluorescent probes can also be incorporated into a protein toprovide for the detection of changes in conformation. Polyethino-adenine, e.g., can be used as a fluorescent nucleotide to measurethe extent of thiolester-zinc finger interaction (see, Karpel (1987) J.Biol. Chem 262:4961).

Finally, a variety of known fluorescent zinc chelators capable ofcomplexing liberated zinc may be used to monitor zinc loss. Bymonitoring the release of zinc from the CCHC zinc finger arrays, theeffect of a given reagent may be determined (Rice (1996) J. Med. Chem.39:3606-3616; Rice (1996) Science 270:1194-1197).

Detection of Disulfide Cross-Linked NC Protein by Gel-Mobility ShiftAssays

Purified concentrated retrovirus and antisera against the purified NCprotein of the virus can be used to test the ability of a specificcompound to penetrate the virus and react with the NC protein by formingdisulfide complexes in the core of the virus. Compound are mixed withthe whole retrovirus under reaction conditions appropriate for thecompound. The virus is then removed from the reagent by centrifugationand disrupted in, e.g., standard SDS-PAGE sample buffer with (reduced)and without (non-reduced) 2-mercaptoethanol. The viral proteins are thenseparated by standard SDS-PAGE and the sample examined for the presenceor absence of the monomeric zinc finger protein in the non-reducedsample. Depending upon the virus used in the experiment and theconditions of electrophoresis, the zinc finger protein can be visualizedby protein staining methods, or by immuno-blot methods. Compounds whichreact with the zinc finger protein by attacking the zinc fingercomplexes and forming disulfide cross-linked products inactivate thevirus. Thus, compounds of interest (i.e., those which causecross-linking), including the thiolesters of the invention, reduce theamount of monomeric zinc finger protein detected. For example,thiolester compound 45 is a strong cross-linker of intravirion NCp7resulting in complete loss of monomeric NCp7 as detected by Westernblotting.

The thiolester-treated virions can also be tested for infectivity. Thevirions are suspended in media (rather than solubilized) and added totarget cells. The cultures are then examined to determine whether thevirions are still active. To determine whether the treated virusparticles are active, the cells are monitored for the presence ofintracellularly-synthesized viral RNA using, for example, the polymerasechain reaction (PCR) (Rice (1993) Proc. Natl. Acad. Sci. USA90:9721;Turpin (1996) J. Virol. 70:6180). Alternatively, cytoprotectionassays can be used (Weislow (1993) J. Natl. Cancer Inst. 81:577).

An example of a compound which can be used as a control in the gelmobility shift assay is azodicarbonamide (ADA), a compound which iscommercially available from the Aldrich Chemical Company (Milwaukee,Wis.). ADA also inactivates HIV-1 virus, as determined using the PCRassay described above.

A kit incorporating the gel-mobility shift concept can be used toidentify and study thiolesters which are able to penetrate intact virusand to induce disulfide cross-links in the viral zinc finger proteins.Such a kit would contain, for example, purified concentrated retrovirusand appropriate size standards to monitor the change in mobility throughthe gel due to disulfide cross-linking.

High Pressure Liquid Chromatography (HPLC) Purified NC Proteins forStructural Characterization of Reaction Products

Highly purified retroviral zinc finger proteins can be produced byexpression from vectors generated through recombinant DNA technology.These proteins when reconstituted with zinc, as described by Summers(1992) Protein Science 1:563-567, provide the source of the NC proteinscontaining the zinc fingers that are the targets for attack by thethiolester compounds of this invention. When the zinc fingers proteinsreact with identified compounds, the reaction produces a covalent changein the zinc finger protein, and the modified protein can be separatedfrom the unreacted protein by, for example, reversed phase HPLC.

The purified proteins and these separation methods are used to obtainsufficient modified protein (i.e., products of the reaction) forchemical and structural analysis. The purified reaction products areisolated and their structures determined by standard N-terminal Edmandegradation. However, for any specific reagent, the gradients and HPLCconditions will depend upon the NC protein and the reaction products.

This procedure is used to identify thiolester compounds which react withHIV-1 zinc fingers. The reaction conditions and HPLC conditions for thedata presented were similar to those described above.

Kits standardizing these techniques may be constructed such that theycontain, for example, purified retroviral zinc finger proteins.

Nuclear Magnetic Resonance-Based Detection of Zinc Loss

NMR can be used to monitor the loss of zinc from retroviral zinc fingerproteins (see, e.g., Rice (1993) Nature 361:473475; McDonnell (1997) J.Med. Chem. 40:1969; Rice (1997) Nature Medicine 3:341-345). It isexpected that one of skill is familiar with the general technique of NMRand its many applications to monitor protein-ligand interactions.Briefly, the atoms in retroviral zinc finger proteins bound to zincshare a different local environment than zinc finger proteins which lackzinc. The difference in local environment leads to distinct NMR spectrafor protein molecules which bind zinc, versus those that do not. Bymonitoring, for example, the proton (¹H) spectrum of a sample containingmetal ion-chelated zinc finger protein and a compound of the presentinvention over time, it is possible to measure whether the compoundcauses the protein to loose its zinc ion.

Since NMR can be used to provide the percent of protein molecules whichare bound to zinc over time, it is also possible to use this techniqueto define the reaction kinetics of a given reaction. Similarly, NMR maybe used to monitor the effect of test compounds upon the binding of zincfinger proteins to nucleic acid complexes. Kits containing e.g.,purified retroviral zinc finger proteins and oligonucleotides may beused to standardize the practice of this method.

Determining Thiolester Anti-Viral Activity

A thiolester is within the scope of the invention if it displays anyantiviral activity (i.e., any ability to decrease or diminish thetransmission of or the replicative capacity of a virus). The antiviralactivity can be determined empirically by clinical observation orobjectively using any in vivo or in vitro test or assay, e.g., the XTTcytoprotection assay (described herein), measuring Tat-induced activity(as in the HeLa-CD4-LTR-beta-gal (MAGI cells) assay and detectingTat-induced beta-galactosidase activity, see, e.g., Tokunaga (1998) J.Virol. 72:6257-6259), and the like. A thiolester with any degree ofmeasurable antiviral activity is within the scope of the invention evenif no metal ion dissociation is detectable.

One exemplary means to determine antiviral activity is with CEM-SS cellsand virus (e.g., HIV-1RF) (MOI=0.01) using the XTT(2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide) cytoprotection assay, as described by Rice (1993) PNAS90:9721-9724, and Rice (1997) Antimicrob. Agents Chemother. 41:419-426.Briefly, cells are infected with HIV-1 RF (or other virus to be tested)in the presence of various dilutions of test compounds (thiolesters andcontrols). The cultures are incubated for seven days. During this timecontrol cultures without protective compounds (i.e., compounds withanti-viral activity) replicate virus, induce syncytia, and result inabout 90% cell death. The cell death is measured by XTT dye reduction.XTT is a soluble tetrazolium dye that measures mitochondrial energyoutput, similar to MTT. Positive controls using dextran sulfate (anattachment inhibitor) or 3′-Azido-2′-3′-dideoxythymidine, AZT (a reversetranscriptase inhibitor) are added to each assay. Individual assays aredone in duplicate using a sister plate method.

Effective antiviral concentrations providing 50% cytoprotection (EC₅₀),and cellular growth inhibitory concentrations causing 50% cytotoxicity(IC₅₀) are calculated.

Alternatively, any virus can be grown in culture, or in an in vivo(animal) model, in the presence or absence of a thiolester or athiolester-containing pharmaceutical formulation to test for anti-viral,viral transmission-inhibiting activity and efficacy. Any virus, assay oranimal model which would be apparent to one of skill upon review of thisdisclosure can be used, see, e.g., Lu (1997) Crit. Rev. Oncog.8:273-291; Neildez (1998) Virology 243:12-20; Geretti (1998) J. Gen.Virol. 79:415-421; Mohri (1998) Science 279:1223-1227; Lee (1998) Proc.Natl. Acad. Sci. USA 95:939-944; Schwiebert (1998) AIDS Res. Hum.Retroviruses 14:269-274.

For in vitro assays, any measurable decrease in the viral load of aculture grown in the presence of a thiolester test compound as comparedto a positive or negative control compound is indicative of ananti-viral, transmission-inhibiting effect. Typically, at least a 30%reduction in viral load observed, generally, between 10% and 99%. Asdiscussed in definition section, above, any relevant criteria can beused to evaluate the antiviral efficacy of a thiolester composition orthiolester-containing formulation.

Cloning and Expression of Retroviral Nucleocapsid Proteins

The novel thiolesters of the invention are capable of dissociating ametal ion from a zinc finger in vitro. Zinc finger containing proteinsare used to detect the dissociation of a metal ion from a zinc fingermotif and in the methods and kits of the invention. General laboratoryprocedures for the cloning and expression of zinc finger motifs andproteins containing these motifs can be found, e.g., current editions ofSambrook, et al., Molecular Cloning A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. Greene Publishingand Wiley-Interscience, New York (1987). Sequences and sources of zincfingers, including nucleic acids, proteins and viral sources, arepublicly available, for example, through electronic databases, such as,e.g., The National Center for Biotechnology Information athttp://www.ncbi.nlm.nih.gov/Entrez/, or, The National Library ofMedicine at http ://www.ncbi.nlm.nih.gov/PubMed/.

The nucleic acid compositions that may be used to express zincfinger-containing proteins, whether RNA, cDNA, genomic DNA, or a hybridof the various combinations, can be isolated from natural sources, ormay be synthesized in vitro. Recombinant DNA techniques can be used toproduce polypeptides. In general, the DNA encoding the polypeptide orpeptide of interest are first cloned or isolated in a form suitable forligation into an expression vector. After ligation, the vectorscontaining the DNA fragments or inserts are introduced into a suitablehost cell for expression of the recombinant polypeptides. Thepolypeptides are then isolated from the host cells. The nucleic acidsmay be present in transformed or transfected whole cells, in atransformed or transfected cell lysate, or in a partially purified orsubstantially pure form. Techniques for nucleic acid manipulation ofgenes encoding zinc finger-containing proteins, such as subcloningnucleic acid sequences into expression vectors, labeling probes, DNAhybridization, and the like are described, e.g., in Sambrook andAusubel, supra.

Once the DNAs are isolated and cloned, one can express the desiredpolypeptides in a recombinantly engineered cell such as bacteria, yeast,insect, or manmalian cells. It is expected that those of skill in theart are knowledgeable in the numerous expression systems available forexpression of the recombinantly produced proteins. No attempt todescribe in detail the various methods known for the expression ofproteins in prokaryotes or eukaryotes will be made. In brief summary,the expression of natural or synthetic nucleic acids encodingpolypeptides will typically be achieved by operably linking the DNA orcDNA to a promoter (which is either constitutive or inducible), followedby incorporation into an expression vector. The vectors can be suitablefor replication and integration in either prokaryotes or eukaryotes.Typical expression vectors contain transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the DNA encoding recombinant polypeptides. Toobtain high level expression of a cloned gene, it is desirable toconstruct expression plasmids which contain, at the minimum, a promoterto direct transcription, a ribosome binding site for translationalinitiation, and a transcription/translation terminator.

Thiolesters as Viricidals

The invention provides a composition comprising a bio-organic or othermaterial and an amount of a thiolester of the invention effective toinactivate any virus (susceptible to inactivation by a thiolester) whichis or may contaminate the material. The material can be bio-organic,such as, e.g., blood plasma, nutrient media, protein, a pharmaceutical,a cosmetic, a sperm or oocyte preparation, cells, cell cultures,bacteria, viruses, foods, drinks. They can be surgical or other medicalmaterials, such as, e.g., implant materials or implantable devices (e.g.plastics, artificial heart valves or joints, collagens), medicalmaterials (e.g., tubing for catheterization, intubation, IVs) andcontainers (e.g., blood bags, storage containers), and the like.Alternatively, a thiolester of the invention can be in the form of acomposition which is applied to any of the above materials as aviricidal reagent and removed before the material's use. The viricidalcomposition can contain a mixture of different thiolesters of theinvention in varying amounts. For example, thiolesters can be added tocell cultures to reduce the likelihood of viral contamination, providingadded safety for the laboratory workers.

Thiolesters as Pharmaceutical Formulations

The invention also provides pharmaceutical formulations comprising thethiolesters of the invention. These thiolesters are used inpharmaceutical compositions that are useful for administration tomammals, particularly humans to for the treatment of viral, especiallyretroviral, infections.

The thiolesters of the invention can be formulated as pharmaceuticalsfor administration in a variety of ways. Typical routes ofadministration include both enteral and parenteral. These include, e.g.,without limitation, subcutaneous, intramuscular, intravenous,intraperitoneal, intramedullary, intrapericardiac, intrabursal, oral,sublingual, ocular, nasal, topical, transdermal, transmucosal, orrectal. The mode of administration can be, e.g., via swallowing,inhalation, injection or topical application to a surface (e.g., eyes,mucous membrane, skin). Particular formulations typically areappropriate for specific modes of administration. Various contemplatedformulations include, e.g., aqueous solution, solid, aerosol, liposomaland transdermal formulations. Details on techniques for formulation andadministration are well described in the scientific and patentliterature, see, e.g., the latest edition of “Remington's PharmaceuticalSciences” (Mack Publishing Co, Easton Pa.).

Aqueous Solutions for Enteral, Parenteral or Transmucosal Administration

Examples of aqueous solutions that can be used in formulations forenteral, parenteral or transmucosal drug delivery include, e.g., water,saline, phosphate buffered saline, Hank's solution, Ringer's solution,dextrose/saline, glucose solutions and the like. The formulations cancontain pharmaceutically acceptable auxiliary substances to enhancestability, deliverability or solubility, such as buffering agents,tonicity adjusting agents, wetting agents, detergents and the like.Additives can also include additional active ingredients such asbactericidal agents, or stabilizers. For example, the solution cancontain sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate or triethanolamineoleate. These compositions can be sterilized by conventional, well-knownsterilization techniques, or can be sterile filtered. The resultingaqueous solutions can be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile aqueous solutionprior to administration.

Aqueous solutions are appropriate for injection and, in particular, forintravenous injection. The intravenous solution can include detergentsand emulsifiers such as lipids. Aqueous solutions also are useful forenteral administration as tonics and administration to mucous or othermembranes as, e.g., nose or eye drops. The composition can contain athiolester in an amount of about 1 mg/ml to 100 mg/ml, more preferablyabout 10 mg/ml to about 50 mg/ml.

Formulations for Enteral or Transdermal Delivery

Solid formulations can be used for enteral administration. They can beformulated as, e.g., pills, tablets, powders or capsules. For solidcompositions, conventional nontoxic solid carriers can be used whichinclude, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10%-95% of activeingredient.

A non-solid formulation can also be used for enteral (oral)administration. The carrier can be selected from various oils includingthose of petroleum, animal, vegetable or synthetic origin, e.g., peanutoil, soybean oil, mineral oil, sesame oil, and the like. See Sanchez, etal., U.S. Pat. No. 5,494,936. Suitable pharmaceutical excipients includestarch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk, glycerol, propyleneglycol, water, ethanol, and the like. Nonionic block copolymerssynthesized from ethylene oxide and propylene oxide can also bepharmaceutical excipients; copolymers of this type can act asemulsifying, wetting, thickening, stabilizing, and dispersing agents,see, e.g., Newman (1998) Crit. Rev. Ther. Drug Carrier Syst. 15:89-142.

A unit dosage form, such as a tablet, can be between about 50 mg/unit toabout 2 grams/unit, preferably between about 100 mg/unit to about 1gram/unit.

Topical Administration: Transdermal/Transmucosal Delivery

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated can be used in theformulation. Such penetrants are generally known in the art, andinclude, e.g., for transmucosal administration, bile salts and fusidicacid derivatives. In addition, detergents can be used to facilitatepermeation. Transmucosal administration can be through nasal sprays, forexample, or using suppositories.

For topical administration, the agents are formulated into ointments,creams, salves, powders and gels. In one embodiment, the transdermaldelivery agent can be DMSO. Transdermal delivery systems can alsoinclude, e.g., patches.

The thiolesters can also be administered in sustained delivery orsustained release mechanisms, which can deliver the formulationinternally. For example, biodegradeable microspheres or capsules orother biodegradeable polymer configurations capable of sustaineddelivery of a composition can be included in the formulations of theinvention (see, e.g., Putney (1998) Nat. Biotechnol. 16:153-157).

Formulation Delivery by Inhalation

For inhalation, the thiolester formulation can be delivered using anysystem known in the art, including dry powder aerosols, liquids deliverysystems, air jet nebulizers, propellant systems, and the like. See,e.g., Patton (1998) Biotechniques 16:141-143; inhalation deliverysystems by, e.g., Dura Pharmaceuticals (San Diego, Calif.), Aradigm(Hayward, Calif.), Aerogen (Santa Clara, Calif.), Inhale TherapeuticSystems (San Carlos, Calif.), and the like.

For example, the pharmaceutical formulation can be administered in theform of an aerosol or mist. For aerosol administration, the formulationcan be supplied in finely divided form along with a surfactant andpropellant. The surfactant preferably is soluble in the propellant.Representative of such agents are the esters or partial esters of fattyacids containing from 6 to 22 carbon atoms, such as caproic, octanoic,lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleicacids with an aliphatic polyhydric alcohol or its cyclic anhydride suchas, for example, ethylene glycol, glycerol, erythritol, arabitol,mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, andthe polyoxyethylene and polyoxypropylene derivatives of these esters.Mixed esters, such as mixed or natural glycerides can be employed. Thesurfactant can constitute 0.1%-20% by weight of the composition,preferably 0.25%-5%. The balance of the formulation is ordinarilypropellant. Liquefied propellants are typically gases at ambientconditions, and are condensed under pressure. Among suitable liquefiedpropellants are the lower alkanes containing up to 5 carbons, such asbutane and propane; and preferably fluorinated or fluorochlorinatedalkanes. Mixtures of the above can also be employed. In producing theaerosol, a container equipped with a suitable valve is filled with theappropriate propellant, containing the finely divided compounds andsurfactant. The ingredients are thus maintained at an elevated pressureuntil released by action of the valve. See, e.g., Edwards (1997) Science276:1868-1871.

A nebulizer or aerosolizer device for administering thiolesters of thisinvention typically delivers an inhaled dose of about 1 mg/m³ to about50 mg/m³.

Delivery by inhalation is particular effective for delivery torespiratory tissues for the treatment of respiratory conditionsincluding an inflammatory component.

Other Formulations

In preparing pharmaceuticals of the present invention, a variety offormulation modifications can be used and manipulated to alterpharmacokinetics and biodistribution. A number of methods for alteringpharmacokinetics and biodistribution are known to one of ordinary skillin the art. For a general discussion of pharmacokinetics, See,Remington's Pharmaceutical Sciences, supra, Chapters 37-39.

Administration

The thiolester of the invention are used in the treatment and preventionof viral infection, particularly, retroviral infections. The amount ofthiolester adequate to accomplish this is defined as a “therapeuticallyeffective dose.” The dosage schedule and amounts effective for this use,i.e., the “dosing regimen,” will depend upon a variety of factors,including frequency of dosing, the stage of the disease or condition,the severity of the disease or condition, the general state of thepatient's health, the patient's physical status, age and the like. Incalculating the dosage regimen for a patient, the mode of administrationalso is taken into consideration.

The dosage regimen must also take into consideration thepharmacokinetics, i.e., the thiolester's rate of absorption,bioavailability, metabolism, clearance, and the like (see, e.g.; thelatest Remington's edition, supra).

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of a thiolester sufficient to treat the patient effectively.The total effective amount of a thiolester of the present invention canbe administered to a subject as a single dose, either as a bolus or byinfusion over a relatively short period of time, or can be administeredusing a fractionated treatment protocol, in which the multiple doses areadministered over a more prolonged period of time. One skilled in theart would know that the concentration of a thiolester of the presentinvention required to obtain an effective dose in a subject depends onmany factors including, e.g., the pharmacokinetics of the prodrug and ofits hydrolysis product, the age and general health of the subject, theroute of administration, the number of treatments to be administered andthe judgment of the prescribing physician. In view of these factors, theskilled artisan would adjust the dose so as to provide an effective dosefor a particular use.

Vaccine Formulations Comprising the Thiolesters of the Invention

The invention also provides an isolated and inactivated virus, where thevirus is inactivated by a method comprising contacting the virus with athiolester compound of the invention, wherein contacting said virus withsaid compound inactivates said virus. In one embodiment the isolated andinactivated virus further comprises a vaccine formulation. A vaccineformulation of the invention can also comprises an isolatedthiolester-complexed viral protein.

The thiolester-complexed, inactivated viruses of the invention are usedin vaccine formulations that are useful for administration to mammals,particularly humans to treat and generate immunity to of a variety ofviral diseases, particularly retroviral infections, such as HIV-1. Thevaccine formulations can be given single administrations or a series ofadministrations. When given as a series, inoculations subsequent to theinitial administration are given to boost the immune response and aretypically referred to as booster inoculations.

The vaccines of the invention contain as an active ingredient animmunogenically effective amount of a thiolester-complexed, inactivated,virus. Useful carriers are well known in the art, and include, e.g.,thyroglobulin, albumins such as human serum albumin, tetanus toxoid,polyamino acids such as poly(D-lysine: D-glutamic acid), influenza,hepatitis B virus core protein, hepatitis B virus recombinant vaccineand the like. The vaccines can also contain a physiologically tolerable(acceptable) diluent such as water, phosphate buffered saline, orsaline, and further typically include an adjuvant. Adjuvants such asincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, oralum are also advantageously used to boost an immune response.

Uses of Thiolester Inactivated Viruses and Thiolester-Complexed Proteins

In addition to uses as vaccines, thiolester-inactivated viruses andthiolester-complexed viral proteins have a variety of uses. For example,thiolester-complexed viral proteins or thiolester-inactivated virusescan be used as reagents for the detection of corresponding anti-viralantibodies. A very commonly used test to determine if an individual isinfected with a virus, such as HIV, is to screen for the presence ofantiviral antibodies. Thiolester-inactivated virion orthiolester-complexed viral protein can be used in these detection testsas trapping antigens or control antigens. See, e.g., Hashida (1997) J.Clin. Lab. Anal. 11:267-286; Flo (1995) Eur. J. Clin. Microbiol. Infect.Dis. 14:504-511.

Thiolester-inactivated virion or thiolester-complexed viral protein canbe used as crystallization reagents for X-ray crystallization analysisor other ultrastructural studies, see, e.g., Yamashita (1998) J. Mol.Biol. 278:609-615; Wu (1998) Biochemistry 37:4518-4526. They can also beused as molecular weight, pI or other controls in various physiochemicalexperiments and methodologies.

Kits and Apparatus

In an additional aspect, the present invention provides kits embodyingthe methods and apparatus herein. Kits of the invention optionallycomprise one or more of the following: (1) a thiolester or thiolestercomponent as described herein; (2) instructions for practicing themethods described herein, and/or for using the thiolester or thiolestercomponent; (3) one or more assay component; (4) a container for holdingthiolesters, assay components, or apparatus components useful formanipulating thiolesters or practicing the methods herein, and, (5)packaging materials.

In a further aspect, the present invention provides for the use of anycompound, apparatus, apparatus component or kit herein, for the practiceof any method or assay herein, and/or for the use of any apparatus orkit to practice any assay or method herein.

Uses of Thiolesters

In a further aspect, the present invention provides for the use of anythiolester composition, virus, inactivated virus or viral component,cell, cell culture, mammal, apparatus, apparatus component or kitherein, for the practice of any method or assay herein, and/or for theuse of any apparatus or kit to practice any assay or method hereinand/or for the use of viruses, cells, cell cultures, compositions orother features herein as a medicament. The manufacture of all componentsherein as medicaments for the treatments described herein is alsoprovided and apparent upon review of the foregoing.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are intended neither to limit or define the invention in any manner.

EXAMPLES Example 1 Capillar Electrophoresis

One exemplary means to determine the metal ion displacing capability ofa thiolester of the invention is through capillary electrophoresis. TheNCp7 protein complexes two zinc ions, each with a formal charge of +2.Reagents that react with the protein and remove the zinc ions cause achange in the conformation and charge of the protein. Thus theelectrophoretic mobility of the reacted protein will differ from themobility of the unreacted protein. Changes in electrophoretic mobilityof the protein can easily be detected by capillary zone electrophoresis(CZE).

The capillary column buffer is 0.001 M sodium phosphate at pH 3.0, andprotein are detected by UV absorption at 215 nm. The sample tubes cancontain about 10 or more microliters of a solution consisting of 0.25micrograms of NCp7 per ml in water at pH 7.0, with or without added zincfinger reactive composition, e.g., a thiolester of the invention. Sampletubes are placed in an automatic sample injector. At programmedintervals 10 μL of sample are drawn into the capillary column and thedata was collected as UV absorption per minute. Unmodified p7NC gives asharp peak of migrating protein passing the detector in about 7.95minutes. Modifications of the protein, caused by reaction with the testcompound of choice, are revealed by a change in this pattern.

Capillary electrophoresis has the advantage of simple automation, sincemany different samples can be loaded into the sample holding rack andanalyzed in successive runs. Each run requires about 10 minutes and eachsample tube can be analyzed many times.

Example 2 Preparation of N-[2-(5-Pyridiniovaleroylthio)benzoyl]-3-aminoprogionamide Bromide (YS1332D)—Templates VI and VII, andN-[2-(5-Pyridiniovaleroylthio)glycinamide Bromide (YS1333D—Templates Vand VII)

Synthesis of YS1332A, a Disulfide Benzamide Intermediate

2,2′-Dithiodibenzoyl chloride (3.43 g, 10 mmol) was added to a clearsolution of β-alanine amide hydrochloride (NOVA Biochem) (3.1 g, 25mmol) and 4-methylmorpholine (NMM; 5 ml, 45 mmol) inN,N-dimethylacetamide (DMA; 30 ml) and water (5 ml) at room temperature(RT). The mixture changed to clear reddish-brown solution in 30 min. Thesolution was stirred at RT for 3 days, during which a precipitate wasformed. The mixture was added to 1M HCl (500 ml). The resultingyellow-orange precipitate was collected, washed with water and driedunder vacuum. Yield was 2.93 g (65%).

YS1333A was synthesized similar to YS1332A, but the reaction time was 1day, not 3 days. Yield was 4 g (95%).

Synthesis of YS1332B, Reduction of YS1332A to a Thiol Intermediate

To the mixture of YS1332A (2 g, 4.5 mmol) in DMF (18 ml) and water (2ml) was added tris(2-carboxyethyl)phosphine hydrochloride (1.5 g, 5.2mmol) and triethylamine (0.5 ml) at RT. The mixture changed to a clearorange solution in 5 min. It was stirred for 1 h, and then added toethyl ether (200 ml). The precipitate was collected, washed with water(3×20 ml) and dried under vacuum. Yield was 1.56 g (77%) of whiteproduct.

YS1333B was made in the same manner as YS1332B. Yield was 1.5 g (76%).

Synthesis of YS1332C, a Haloalkanoyl Thioester Intermediate

To a solution of YS1332B (0.8 g, 3.6 mmol) in DMA (5 ml) was added5-bromovaleryl chloride (1.4 ml, 10.8 mmol) at RT under nitrogen. Themixture was stirred for 1 h, then added to ethyl ether (80 ml). Theprecipitate was collected and dissolved in DMF (5 ml). The solution wasadded to 10% sodium bicarbonate (40 ml; pH=8) with stirring. The whiteprecipitate was collected, washed with water and dried. Yield was 0.83 g(70%).

YS1333C was made the same way as YS1332C. Yield was 1.27 g (74%).

Synthesis of YS1332D, a Pyridinioalkanoyl Thioester (PATE)

A solution of YS1332C (0.2 g, 0.52 mmol) in pyridine (4 ml) was stirredat RT under nitrogen overnight. The mixture was added to ethyl ether (40ml). The white precipitate was collected, washed with ether and dried.Yield was 0.23 g (95%).

YS1333D was made in a similar manner to YS1332D, but the reactionmixture was stirred for 2 days, not 1 day. Yield was 0.22 g (90%).

Example 3 Preparation ofN-[2-(5-Pyridiniovaleroylthio)benzoyl]-3-aminopropionic Acid Bromide(YS1334D)—Templates VI and VII, andN-[2-(5-Pyridiniovaleroylthio)benzoyl]-L-isoleucine Bromide(YS1324D)—Templates V and VII

Synthesis of YS1334A, a Disulfide Benzamide Intermediate

2,2′-Dithiodibenzoyl chloride (3.43 g, 10 mmol) was added to a clearsolution of β-alanine t-butyl ester hydrochloride (NOVA Biochem) (4.8 g,25 mmol) and 4-methylmorpholine (NMM; 5 ml, 45 mmol) inN,N-dimethylacetamide (DMA; 10 ml) at room temperature (RT). The mixturewas stirred at RT overnight. To it was added heptane (200 ml). Theresulting precipitate was dissolved in a small amount of DMF, then addedto 1M HCl (500 ml). The viscous solid precipitate was collected, washedwith water and dried under vacuum. Yield was 5.1 g (91%)

YS1322A was synthesized in the same way from 5.0 mmol of startingdichloride and an excess of L-isoleucine t-butyl ester hydrochloride.Yield was 3.10 g (65%) of the disulfide.

Synthesis of YS1334B, Reduction of YS1334A to a Thiol Intermediate

To a solution of YS1334A (1.8 g, 3.2 mmol) in DMF (9 ml) and water (1ml) was added tris(2-carboxyethyl)phosphine hydrochloride (1.3 g, 4.5mmol) and triethylamine (0.45 ml) at RT. The mixture was stirred for 1h, and then added to 0.5 M HCl (200 ml). The viscous residue wascollected, washed with water and dried under vacuum. Yield was 1.24 g(69%).

YS1324A was made in the same way as YS1334B. 2.0 g of disulfide yielded1.5 g of thiol (75%).

Synthesis of YS1334C, a Haloalkanoyl Thioester Intermediate

To a solution of YS1334B (1.24 g, 4.4 mmol) in DMA (5 ml) was added5-bromovaleryl chloride (1.5 ml, 11 mmol) at RT under nitrogen. Themixture was stirred for 1 h, then added to ethyl ether (100 ml). Thesolvent was evaporated to 5 ml, and the resulting viscous residue wasseparated, washed with 10% sodium bicarbonate (20 ml; pH=8) and water(40 ml), and dried. Yield was 1.6 g (82%).

YS1324B was prepared from 1.0 g of the thiol to yield 0.95 g of product(65% yield).

Synthesis of YS1334D, a Pyridinioalkanoyl Thioester (PATE)

A solution of YS1334C (1.6 g, 3.6 mmol) in pyridine (10 ml) was stirredat RT under nitrogen overnight. It was then added to a solution of ethylether (200 ml) and heptane (100 ml). The precipitated phase wasdissolved in methanol (5 ml) and added to a solution of ether (80 ml)and heptane (200 ml). The precipitate was dissolved in a solution oftrifluoroacetic acid (10 ml) and formic acid (3 g). The solution wasstirred at RT overnight and was reduced to 5 ml with a stream ofnitrogen. To the residue was added ethyl ether (40 ml). The precipitatewas collected and dried. Yield was 0.9 g (53%).

YS1324D was prepared from 0.60 g of the corresponding bromovaleroylthioester to yield 0.58 g (58%) of the isoleucine-bearing PATE.

The foregoing is offered for purposes of illustration. It will bereadily apparent to those skilled in the art that the materials,procedural steps and other parameters of the methods and kits describedherein may be further modified or substituted in ways without departingfrom the spirit and scope of the invention.

1. A method for inactivating a retrovirus comprising a proteincomprising a zinc finger, said method comprising contacting saidretrovirus with a compound having the formula:

wherein: R₁ is Y—Z; Y is —(CH₂)_(m)—, wherein m is an integer from 1 to6; Z is pyridinio having the structure:

wherein R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, andR₁₆, are-H; and R₉ is (O═S═O)-G′, wherein G′ is selected from the groupconsisting of —NH₂,—NH-alkyl, —NH-acyl groups, nitroaryl, andaryl-NH-acyl; wherein contacting said retrovirus with said compoundinactivates said retrovirus.
 2. The method of claim 1, wherein saidcompound administered to inhibit the transmission of the retrovirus. 3.The method of claim 1, wherein said compound is administeredintra-vaginally or intra-rectally to inhibit the transmission of theretrovirus.
 4. The method of claim 1, wherein said compound isadministered to a human as a pharmaceutical formulation.
 5. The methodof claim 1, wherein the contacting of said retrovirus with said compoundis performed in a blood product, blood plasma, nutrient media, protein,a pharmaceutical, a cosmetic, a sperm or oocyte preparation, cells, cellcultures, bacteria, viruses, food or drink.
 6. The method of claim 1,further comprising contacting said retrovirus with a non-thiolesteranti-retroviral agent selected from the group consisting of zidovudine,dideoxycytidine 5-triphosphate and didanosine.
 7. The method of claim 1,wherein the retrovirus is selected from an HIV-1, an HIV-2, an SIV, aBIV, an EIAV, a Visna, a CaEV, an HTLV-1, a BLV, an MPMV, an MMTV, anRSV, an MuLV, a FeLV, a BaEV, or an SSV retrovirus.
 8. The method ofclaim 1, wherein said retrovirus is an HIV-1.